1. health and fitness
2. disease
3. cancer

GLIIFCA 21
September 28th – September 30th, 2012
Buffalo, NY, USA
www.gliifca.org
2011 Program Chairs:
Site Organizer:
David Hedley, President
Michael Sramkoski
Amanda (Nicole) White
Alexander Nakeff
Corporate Sponsors & Members:
GLIIFCA 21
Program
Table of Contents
General Information…………………….….2
General Schedule……………………………5
Speaker Schedule – At a Glance…………...6
Industrial Science Symposium..……………7
Symposium I Schedule and Abstracts….... 14
Keynote Address Abstract ……………. 17
Luncheon Roundtable Topics ………….. 18
Symposium II Schedule and Abstracts….. 19
Symposium III Schedule and Abstracts….22
Poster Abstracts…………………………... 25
Steering Committee Info…………………. 36
List of Attendees………………………….. 37
Registered Vendor Representatives……... 39
Blank page for notes……...………………. 41
1
GLIIFCA 21 GENERAL INFORMATION
(What You Always Wanted To Know About GLIIFCA 21 But Were Afraid To Ask!)
CONFERENCE REGISTRATION DESK: Lobby
Conference registration fee includes Friday reception, Sat/Sun breakfast, Sat. Lunch, evening
reception, banquet and coffee breaks.
Friday, Sept 28
Saturday, Sept 29
Sunday, Sept 30
6:00 to 11:00pm
8am to 11pm
9am to 12:30pm
POSTERS:
SET UP: Friday, September 28 after 12 noon in 106A&D
Νumbers on posters correspond to poster abstract numbers in the program
Poster board size= 3 ft wide and 4 ft high
Please mount one poster on each side of a poster board using VELCRO only
Viewing on Friday to Sunday 10:30am
Presentation and Judging: Saturday 5:00 to 7:30pm
EXHIBITS:
Scheduled exhibitors will have booths in the Exhibit/Poster area (106A&D)
Βooths will be open from 5:30pm Friday to the end of the meeting
Αll activities other than the plenary sessions, roundtable luncheon, Steering Committee meeting and
banquet
will be located in the Exhibit/Poster area (106A&D)
Please frequent the booths and show our appreciation for the generous financial support provided
by
the exhibitors who substantially help "pay the freight" for this meeting
BREAKFASTS:
Free continental breakfast provided for all registrants in the Exhibit/Poster area (106A&D) on:
Saturday:
7 to 8am
Sunday:
8 to 9am
Steering Committee breakfast meeting, Sunday morning (7:30 to 9am) in 106C
COFFEE BREAKS:
Snacks and drinks available in the Exhibit/Poster area (106A&D) - no need to line up!
2
INDUSTRIAL SCIENCE SYMPOSIUM & FRIDAY RECEPTION:
Industrial Science Symposium presentations: 7 to 9:30pm, 106B
Reception:
5:30 to 8pm and 10 to 11pm, 106A&D
Use 3 drink tickets for wine and beer
SYMPOSIA LOCATION: 106B
SATURDAY LUNCHEON ROUNDTABLES (12 to 1:30pm):
Free lunch/pop; 2/3 ham/turkey and 1/3 veggie in 102 (Tables 1-3), 103 (Tab 4-6), 104 (Tab. 7-10), 105
& Boardroom for non-patricpants in workshops),
Pick up box lunch (roast beef, turkey or veggie) and soft drink displayed in each room and move to
roundtable of 10 labeled with the title of discussion topic - attendance at each table determined from sign
up sheet at GLIIFCA registration desk. Non-participants can lunch in the Boardroom.
SATURDAY WINE AND CHEESE HAPPY HOUR:
5:00 to 8pm in the Exhibit/Poster area (106A&D) with cheese and fresh fruit trays (use
drink tickets).
BANQUET:
Free to registrants and paid guests
Commences at 8pm, Ballroom.
Numerous food stations (salad, entrée and dessert) provided to minimize waiting time
Full service bar available for drinks (use drink tickets or pay cash)
DJ with dance music until 11pm; requests encouraged (get up and have fun!)
DRINKS:
Full service bar located in the Exhibit/Poster area (106A&D) for Friday reception and Saturday
afternoon for Wine & Cheese reception and Ballroom for banquet Saturday evening
Three free drink tickets/registrant for beer and wine only – beer in bottles/cans
Mixed drinks – cash bar (your cost).
Αll pop in bottles/cans is free
FACILITIES/SERVICES:
Μessage Board: on easel next to the GLIIFCA Registration Desk, Foyer
Xerox copying, faxing, etc: Ask at BNCC main desk
3
CMLE CREDITS:
To receive CMLE credit, sign application form at the GLIIFCA registration desk
NAME TAGS AND EVALUATION FORMS: Before leaving….
Fill out evaluation form and leave at Registration/Check Out Desk with your name tag
ADDITIONAL ENQUIRIES:
Contact Dr. Alexander Nakeff (Email: [email protected]; Cell: (313) 820-6227) or leave
message for him at the GLIIFCA registration desk
4
GENERAL MEETING SCHEDULE:
All symposia to be held in the Room 106B. Poster viewing, Vendor Exhibits, breakfasts and breaks will all be
held in the Rooms 106A&D.
Friday, September 28th
10a – 4p Resource Managers Workshop
Room 106C
Sponsored by: GLIIFCA & ISAC
Sponsored by: Life Technologies,
Stratedigm, Beckman Coulter, & Verity
Software House
5p – 8p
5p – 10p Registration
Lobby
Poster Presentations and
Judging
8p – 11p GLIIFCA Annual Banquet &
Awards Ceremony
Ballroom
6p – 11p Opening Reception
Rooms 106A&D
Sponsored by: iCyt
7p – 10p Industrial Science Symposium
Room 106B
Party Theme: “Casino Night”
Costume Prize Sponsored by TreeStar
Alex Nakeff Young Investigator Award
Poster Awards
Sponsored by:
R&D Systems (2), Life Technologies (1),
BD Biosciences (1)
Saturday, September 29th
7a – 8a
Continental Breakfast
8:15a – 11:00a Symposium I
Monitoring Immune Function
Travel Stipend Awards
Sponsored by:
R&D Systems (2), BD Biosceinces (1),
Miltenyi Biotec (1), eBioscience (1)
9:30 – 10a
Coffee Break
Sponsored by: DeNovo Software
The Carleton and Sigrid
Stewart Keynote Lecture
11a –12p
12-1:30p
Luncheon Roundtables
Rooms 102-104, 105 & Boardroom for nonparticipants
1:30p – 4:15p Symposium II
Emerging Applications of
Translational Cytometry
3:00p – 3:30p Coffee Break
Sponsored by: Spherotech, Inc.
5:00p – 8p
Sunday, September 30th
8a – 9a
Continental Breakfast
7:30a – 9a
Steering Comm. Meeting
Room 106C
9a – 11:45a
Symposium III
Rare Event Detection
by Flow Cytometry
10:30a – 11a Coffee Break
Sponsored by: Cytek Development
Wine & Cheese Reception
11:45a–12p
Closing Remarks
BEFORE LEAVING: Please return evaluation form and name tag holder to Registration/Check
out Desk. Thank you!
5
Speaker Schedule – At a Glance
Resource Managers’ Workshop
Friday, September 28th, 10:00a – 4:00p
Convener: Sally Quataert
9:45-10:15 Coffee Break
10:15-11:00 Sharon Evans, RPCI, IL-6 – Friend
or Foe? Targeting the Immune
Microenvironment for Cancer
Immunotherapy.
Industrial Science Symposium
Friday. September 28th, 7:00p – 10:00p
Convener: Karen Domenico
7:00-7:20 Mark Munson, Verity Software House.
Data Modeling: A Preferred Analysis Method
for High-Dimensional Flow Cytometry Data.
7:20-7:40 Susan Reynolds, eBioscience, Inc. Using
the Green Laser to Enhance Multi-Colored
Flow Cytometry Experiments.
7:40-8:00 Darin Fogg, Amnis Corporation.
Optimizing the Amnis ImageStreamX for CTC
and other Rare Cell Analysis Applications.
8:00-8:20 Janet Horta, BD Biosciences. BD
FACSJazzTM Cell Sorter Overview – A New
Era in Cell Sorting.
8:20-8:40 Matthew Hsu, EMD Millipore. Cross
Platform Solutions for Autophagy Detection
using the Guava 8HT and Amnis
ImageStream MarkII.
8:40-9:00 Lisa Nichols, Cytek Development. Back
to the future: Updating your cytometer with
affordable custom configurations.
9:00-9:20 Matthew Alexander, Sony iCyt.
Microfluidics Cell Sorting Chip Platform.
9:20-9:40 Barbara Seredick, Life Technologies.
Novel Apoptosis and ROS reagents from
Molecular Probes®.
9:40-10:00 Shane Oram,Miltenyi Biotec, Inc.
Search Deeper: Utilization of the
MACSQuant® Analyzers for analysis of
subcellular particles and microvesicles.
11a-12p The Carleton and Sigrid Stewart
Keynote Lecture.
Luncheon Roundtables
12:00p – 1:30p
Conveners: Paul Champoux
Symposium II – Emergin Applications of
Translational Cytometry
Saturday, September 29th, 1:30p – 5:00p
Convener: Michael Sramkoski
1:30 – 2:15 Orla Macquire, Buffalo. Image
Cytometry Cytometry Based Detection of
Aneuploid by FISH-IS.
2:15 – 3:00 Robert Thacker, Cincinnati
Children’s. Characterizing the crosspresentation of cell-associated antigens by
merocytic dentric cells, a novel DC subset.
3:00-3:30
Coffee Break
3:30 – 4:15 Maria Watson, Universtiy of Toronto.
Development of flow cytometry techniques
for diagnosing and monitoring the epigenetic
status of leukemia patients.
5:00p – 7:30p Poster Presentation and Judging
Symposium III – Rare Event Detection
Sunday, September 30th, 9:00a – 11:45a
Convener: Amanda (Nicole) White
9:00 – 9:45 Alison Allan, London ON, From the
bench to the bedside: image and flow
cytometry analysis of circulating tumor cells.
9:45 – 10:30 Anna Porwit, Toronto General
Hospital. Minimal residual disease in acute
myeloid leukemia. Quantitation by Flow
Cytometry: Why, How, and When?
10:30 – 11:00
Coffee Break
11:00 – 11:45 Vera Donnenberg, University of
Pittsburgh. Cancer Stem Cells: a cell state or
a cell type?
Symposium I – Monitoring Immune Function
Saturday, September 29th, 8:15a – 10:45a
Convener: David Hedley (GLIIFCA 2012,
President)
8:15-9:00 Hans Minderman, RPCI. Monitoring
clinical pharmacodynamics response by image
cytometry.
9:00-9:45 Sally Quataert, University of Rochester,
Immune Monitoring: Development and
Validation of Multichromatic Panels for
Clinical Translational Studies.
6
Industrial Science Symposium
Friday, September 28th
7:00pm – 10:00pm
Room 106B
Convener: Karen Domenico, Children’s National Medical Center
7:00-7:20 Data Modeling: A Preferred Analysis Method for High-Dimensional Flow
Cytometry Data.
Mark Munson, Verity Software House.
7:20-7:40
Using the Green Laser to Enhance Multi-Color Flow Cytometry
Experiments.
Susan Reynolds, eBioscience, Inc.
7:40-8:00
Optimizing the Amnis ImageStreamX for CTC and other Rare Cell Analysis
Applications.
Darin Fogg, Amnis Corporation.
8:00-8:20 BD FACSJazzTM Cell Sorter Overview – A New Era in Cell Sorting.
Janet Horta, BD Biosciences.
8:20-8:40 Cross Platform Solutions for Autophagy Detection using the Guava 8HT
and Amnis ImageStream MarkII.
Matthew Hsu, EMD Millipore.
8:40-9:00
Back to the future: Updating your cytometer with affordable custom
configurations
Lisa Nichols, Cytek Development.
9:00-9:20
Microfluidic Cell Sorting Chip Platform.
Matthew Alexander, Sony iCyt.
9:20-9:40
Novel Apoptosis and ROS reagents from Molecular Probes®.
Barbara Seredick, Life Technologies.
9:40-10:00 Search Deeper: Utiliazation of the MACSQuant® Analyzers for analysis of
subcellular particles and microvesicles.
Shane Oram, Miltenyi Biotec, Inc.
7
Abstracts:
Data Modeling:
A Preferred Analysis Method for High-Dimensional Flow Cytometry Data
Mark Munson
Verity Software House
The concept of data modeling is not new; you've actually been doing it in one dimension for
years. What is new is the extension of modeling principles to multi-measurement space. We
will begin with a familiar example, and then show you how to enter the realm of ndimensional modeling step-by-step, one measurement at a time. You will see how each
additional measurement brings clarity to the analysis, not complexity. You may be surprised
by how much the cells have to tell us - if we will only let them speak, and not muffle them
with gating.
Using the Green Laser to Enhance Multi-Color Flow Cytometry
Experiments
Susan Reynolds
Field Application Scientist, eBioscience, Inc.
Green (532 nm) and yellow-green (561 nm) lasers are increasingly common on today’s flow
cytometers. In addition to being more powerful, these lasers also allow for optimal excitation
of PE and PE tandem dyes and fluorescent reporter proteins. Learn how to successfully
incorporate the use of these lasers in your experiments. Topics to be covered include:
choices of dyes, optimization of filter sets, fluorochrome performance, and compensation
issues.
Optimizing the Amnis ImageStreamX for CTC and other Rare Cell
Analysis Applications
Darin Fogg, Ph.D.
Sales Manager, Eastern Region, Amnis Corporation
The ImageSreamX has been widely adopted for the analysy of CTCs and other rare cells.
Such applications place stringent demands on instrument speed, sample handling efficiency,
and real-time data analysis capabilities. This talk will describe a range of optimizations to the
ImageStreamX that have greatly increased its utility for rare cell analysis and numerous other
applications.
8
BD FACSJazzTM Cell Sorter Overview – A New Era in Cell Sorting
Janet Horta
Senior Product Manager, BD Biosciences
The BD FACSJazz cell sorter incorporates design features that simplify operation of streamin-air cell sorters, to increase the operational efficiency of labs with high workloads, making
the most commonly used sorting applications accessible to researchers with limited flow
cytometry experience. The key features include factory-optimized settings, intuitive
alignment, real-time video monitoring, and BD FACS™ Accudrop technology. Requiring
less than 2 x 2 ft (20 x 20 in, 51 x 51 cm) of bench space, the BD FACSJazz is an easy fit in
a core facility or an individual lab. The power supply, electronics, and fluid tanks are placed
below the lab bench to allow the instrument to occupy a reduced footprint or be installed
easily in a biological safety cabinet.
The BD FACSJazz can be configured with up to three lasers and eight parameters to support
application requirements for individuals and core labs. In a core lab, the BD FACSJazz can
offload sorting demand and free high-end sorters by handling routine applications such as
cloning. The system has also been designed to meet the needs of individuals for applications
such as single-cell analysis, which are driving the accelerated pace of genomics and
nextgeneration sequencing. To simplify setup and training, the BD FACSJazz runs with
factory-optimized settings. It comes with BD FACS™ Sortware, an innovative software
application specifically designed for comprehensive instrument control during acquisition,
sorting, and analysis. With dependable BD performance, a benchtop fit and an affordable
price, the BD FACSJazz™ signals a new era in cell sorting.
Cross Platform Solutions for Autophagy Detection using the Guava 8HT
and Amnis ImageStream MarkII
Matthew Hsu
R&D Director, EMD Millipore
Autophagyis an intracellular catabolic pathway which causes cellular protein and organelle
turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and
Crohn's disease, in addition to aging. It is a tightly regulated process that plays a normal part
in cell growth, development, and cellular homeostasis. Autophagy functions as a
housekeeping mechanism through disposal of aging and dysfunctional proteins and
organelles by sequestering and priming them for lysosomal degradation. Increasing evidence
suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly
influence general cell health. Malfunctions of autophagy can adversely impact longevity and
the capability of cells to function at full capacity.
Here, we describe two key platforms paired with a novel assay design which will allow for
the study of autophagy by flow cytometry. Guava 8HT systems have the capabilities of
higher throughput sampling utilizing a 96-well format and smaller sample sizes for
acquisition. And when combined with the InCyte software module for data analysis and
curve fitting algorithms for small molecule analysis, one can now screen autophagy inducers
or inhibitors more efficiently. Amnis’s ImageStream system is a perfect complementary
platform as it combines the capabilities of microscopy and flow cytometry in a single
platform for quantitative image based cellular analysis of autophagy.
9
Back to the future:
Updating your cytometer with affordable custom configurations
Lisa Nichols
Western Regional Director, Cytek Development
Discover how our upgrade options can extend the useful life of your existing technology by
adding capabilities and updating components. Cytek’s lines of cytometer upgrades are
designed specifically for BD FACScanTM, FACSortTM and FACSCaliburTM flow cytometers.
Extra Parameter (xP) and Digital Extra Parameter (DxP) customizable configurations and
add-on options support a wide range of applications. By leveraging the use of existing proven
fluidics, xP and DxP systems are a cost effective alternative to buying a new cytometer. In
this workshop, Cytek will present various configuration options and demonstrate
experimental data collected using Cytek’s acquisition software, FlowJo Collector’s Edition.
Comparison QC and Biological data from multiple cytometer platforms will be presented.
See for yourself how a Cytek upgrade compares to buying new in both quality and cost and
how an affordable custom configuration allows you to purchase an instrument tailored to
your applications.
Microfluidic Cell Sorting Chip Platform
Matthew Alexander
Global Marketing Manager, Sony iCyt
This session will describe a novel cell sorter platform which utilizes a microfluidic cell
sorting chip. This innovative approach enables a unique cell sorter system that is capable of
automated chip loading as well as automated optical alignment and a sorting parameters
setup. The microfluidic cell sorting chip is manufactured using an industrial plastic material
and a highly precise injection molding duplication. By using optical disc manufacturing
process, the chip has low-cost and high analyzing and sorting performance. In this tutorial,
we investigate a disposable microfluidic cell sorting chip for a sense in channel sorter.
We will also discuss the sy3200 platform. This high end sorter is differentiated with true
innovation in optics, electronics, and industrial design. Sony technology enables fast
acquisition and sorting speeds while ensuring superior sensitivity and resolution. Additional
topics will focus on the sy3200 unique capability to efficiently expand to a dual independent
sorter system as well as the system’s innovative approach to ensuring the highest levels of
biosafety.
Search Deeper: Utilization of the MACSQuant® Analyzers for analysis of
subcellular particels and microvesicles
Shane Oram & Ashley Weant
Flow Cytometry Specialist, Miltenyi Biotec, Inc.
The use of flow cytometry as a technology for small particle analysis has increased in a
growing number of fields, including microbiology and aquatic ecology. As most flow
cytometers are generally designed for analysis of human blood samples and equally sized
particles (~2 µm – 30 µm), some unique challenges arise when analyzing particles in the size
range of 1 µm down to tens of nanometers. In this presentation, set up of the MACSQuant®
Analyzer for analysis of such particles as mitochondria, viruses and microvescicles is
discussed including the use of SSC or fluorescence as the trigger parameters.
10
Novel Apoptosis and ROS reagents from Molecular Probes®
Barbara Seredick
R&D Scientist, Flow Cytometry Systems, Life Technologies
Multicolor flow cytometry provides answers to complex cell biology questions from
population analysis to rare event detection of stem cells. Higher-plexed multicolor flow
cytometry experiments reveal more information at the single cell or population level in less
time. Life Technologies™ offers researchers many reagents and assays to accelerate the
ability to analyze cell function far beyond immunophenotyping, including viability kits, dead
cell stains, and reagents to study apoptosis, oxidative stress, membrane potential, cell cycle,
metabolic activity, and phagocytosis. The CellEvent™ Caspase-3/7 Green Detection
Reagent and the CellROX™ Reagents for oxidative stress detection are two new product
lines that make it easier for researchers to study cell health. The CellEvent Caspase 3/7
Green Detection Reagent is a novel fluoregenic substrate that emits green fluorescence in the
presence of activated caspase 3/7, a hallmark of programmed cell death. This reagent doesn’t
rely on fixation and permeabilization, making it compatible with live cells and permits easy
multiplexing with additional reagents for a more complex analysis of samples.
The
CellROX™ Deep Red reagent is a bright and stable ROS sensor that fluoresces in response
to reactive oxygen species. Also compatible with live cells, CellROX™ offers significant
advantage over existing ROS sensors because it can survive formaldehyde fixation and emits
fluorescence in the far red region of the spectra. In this presentation we will cover use of
these reagents with practical examples and details.
Bringing cell sorting to your benchtop: reliable, affordable cell sorters for
the core lab or your lab
David Coder
Bay bioscience
In the past, high speed cell sorting has been difficult for non-specialist. That is, difficult to
operate reliably, difficult to maintain, and certainly difficult to purchase and keep running.
The new Bay bioscience sorters–the JSAN and the Jr/Swift–provide alternatives that are
reliable, easy to use, easy to maintain, and are very cost effective. A wide range of lasers is
available. The sense-in-quartz flow cell is easily removed for routine cleaning; flow cell
replacement does not require extensive optical re-alignment. All components are easily
accessible by the user. Systems are completely self-contained with an internal vacuum pump
and compressor: Only a single wall socket is needed. Setup for sorting is rapid (less than 15’
from a cold start) and simple. While sorting, the drop delay is monitored and adjusted and the
system can detect changes in stream dynamics to maintain optimal sort conditions. Sorting
into tubes or microtiter plates is supported and you can switch between the two in 5 minutes.
High speed sorting can be done effectively at 30psi, increasing the viability of fragile cell
types. The entire systems are compact, and can fit inside smaller, less expensive Baker
biosafety cabinets further decreasing the costs of sorting with Class 2a biosafety conditions.
Overall, Bay bioscience enables, flexible high speed sorting for the non-specialist: Get more
for your instrument budget!
11
Advanced Models of Spectral Unmixing
David Novo
President, DeNovo Software
Proper software compensation is an integral component of successful polychromatic flow
cytometry. It is also a continual source of confusion among novice (and experienced) users
and requires considerable effort to perform and optimize. Even when applied properly
compensation often results in highly non-intuitive results, such as negative fluorescence and
excessive spreading of dim populations, that are very difficult to understand and interpret in
light of the underlying biology. Much of these artifacts are a result of applying simplistic
mathematical models to convert the observed fluorescence signal into a value proportional to
dye abundance. This talk will discuss more advanced models of spectral unmixing and how
they compares to standard compensation and apply to flow cytometry. Proper application of
these techniques minimizes, and often eliminates, compensation artifacts and in addition can
provide several new metrics for assessing of the quality of the unmixing.
Multiple Flow Cytometric Approaches for Studying DNA Damage
Pathways and Assessing the Level of the Cellular Responses to DNA
Mark Santos, Wenying Zhang, Angelica Olcott, Jason Whalley, Patrick Schneider,
and Matthew Hsu
EMD Millipore
Investigation of DNA damage pathways and assessment of the DNA damage levels in cells
are extremely important for the study of cancer and developing anti-cancer drugs, as defects
in DNA damage pathways can cause cancer and lead to genetic instability and ultimately
uncontrolled cell growth. The activation of the ATM dependent DNA damage pathway by
various agents is carefully monitored and confirmed using a group of directly conjugated and
optimized phospho-specific antibodies against ATM, SMC1 and Histone H2A.X targets; then
the level of DNA damage is quantitatively measured using a pair of antibodies against the
total and phosphorylated H2A.X, in order to detect target-specific of phosphorylation
especially in a mixed cell population. These and other related assays were successfully used
in the evaluation of DNA damaging and anti-neoplastic agents, as well as their impact on cell
cycle, which helped elucidate the mechanisms of cell proliferation, apoptosis, and DNA
repair, ultimately advancing the study for cancer research.
12
Detection of Mouse CD4+ Th Cell Differentiation
by Flow Cytometry and Multiplex
Mark Santos, Yuko Williams, Don Weldon, Wen-Rong Lie, Angelica Olcott, Jason Whalley,
Patrick Schneider, and Matthew Hsu
EMD Millipore
Naïve CD4+ T cells can give rise to a variety of subsets of Th cells depending on the nature
of the immune response, and subsequently release a distinct subset of cytokines.
Conventionally these cytokines can be measured in a secreted format (e.g. Luminex or
ELISAs), but here we describe a novel method to reliably differentiate mouse CD4+ T cells,
and further characterize the differentiated lineages by measuring intracellular cytokines using
flow cytometry. In addition, we also describe the use of a proprietary fixable viability dye in
order to eliminate the false positives associated with non-specific staining.
Based on an optimized protocol for differentiating CD4+ T cells, we have developed
differentiation tools designed to obtain the desired Th cell lineages. Following naïve T cell
differentiation toward specific Th lineages, we further developed a flow cytometry assay to
measure intracellular cytokine production by blocking the secretion of the cytokine with
Brefeldin A. We further eliminate nonspecific staining due to dead or dying cells using a
proprietary fixable viability dye and accurately determine the percentage of Th lineages using
guava bench top flow cytometer and InCyte analysis software.
13
Symposium I
Saturday, September 29th
8:15am-11:00am
Monitoring Immune Function
Room 106B
Convener: David Hedley (GLIIFCA 2012, President)
8:15 – 9:00
Clinical Applications of Imagestream Cytometry
Hans Minderman, Roswell Park Cancer Institure
9:00 – 9:45
Immune Monitoring: Development and Validation of Multichromatic
Panels for Clinical Translational Studies.
Sally A. Quataert, University of Rochester
9:45 – 10:15
Coffee Break (Sponsored by DeNovo Software) – Posters and Exhibits
10:15 –11:00 IL-6 - Friend or Foe? Targeting the Immune Microenvironment for
Cancer Immunotherapy
Sharon S. Evans, Roswell Park Cancer Institute
Abstracts:
Clinical Applications of Imagestream Cytometry
Hans Minderman
Roswell Park Cancer Institute, Buffalo, NY
The ImageStream is a flow cytometry-based image analysis platform that acquires up to 12
spatially correlated, spectrally-separated images of cells in suspension at rates of up to 1000
cells/sec. By combining the high throughput and multiparameter capability of flow cytometry
with the high image content information of microscopy it enables quantitative image analysis
in immunophenotypically defined cell populations in statistically robust cell numbers. One
area of its clinical application is in the study of cellular signal transduction pathways for
which the intracellular localization of signaling intermediaries correlate with activity.
Examples of this will be presented with regards to the cytoplasmic to nuclear translocation of
NFAT1 and p65 upon activation of the NFAT and NFkB transcription factor pathways,
respectively. Examples will be presented how these assays can provide determinants of
response to immunosuppressive and anti-leukemic therapies.
Correspondence: [email protected]
Immune Monitoring: Development and Validation of Multichromatic
Panels for Clinical Translational Studies.
14
Sally A. Quataert, Ph.D., RQAP-GLP
Rochester Human Immunology Center Core Laboratory,
University of Rochester, Rochester, NY
Multichromatic flow cytometry technology provides the opportunity for increasing depth of
analysis and discovery in clinical translational research. However, development of validated
panels capable of giving scientifically sound results for a study across different individuals,
time points, reagent lots and instruments is challenging. Often clinical studies require
following the immune status of subjects over a period of years rather than weeks as in nonclinical animal studies. The cost can also be prohibitive in terms of both reagents and
instrument time. The Rochester Human Immunology Center has worked with investigators
in multiple studies to develop and validate immune monitoring panels that can be readily
adapted to other projects to lower the cost of initial set up. The panels include T, B, natural
killer (NK), dendritic cell (DC), T regulatory phenotyping and intracellular cytokine staining
using 12 to 16-colors in a micro-method minimizing cost while maintaining precision and
optimal staining. Activation markers are accessed in the development phase in order to
insure that direct ex-vivo as well as in vitro stimulation is detectable in rare populations.
Strategies for validation and quality control of results over the study include standardization
of sample handling, use of fluorescent beads, instrument calibration, cryopreserved assay
controls, use of FMO and isotype controls, and careful titration and qualification of new lots
of reagents. When control methods are designed into the flow cytometry panels employed in
a clinical study, it is possible to achieve consistent and comparable results over the entire
course of the study. Examples from a pediatric study for stability of populations during
processing of cord blood specimens, a lymphoma study and an infectious disease study will
be used to illustrate the development process.
Correspondence: [email protected]
IL-6 - Friend or Foe?
Targeting the Immune Microenvironment for Cancer Immunotherapy
Sharon S. Evans, Ph.D.
Department of Immunology,
Roswell Park Cancer Institute, Buffalo, NY
Efforts to harness the exquisite specificity of cytolytic CD8 T lymphocytes to destroy tumors
have had minimal clinical success, in part because the mechanisms impeding cancer
immunity are incompletely understood. We have recently identified restricted homeostatic
trafficking of CD8 T cells across tumor-vascular gateways as a rate-limiting step during the
effector phase of tumor immunity. In contrast to the protumorigenic activity commonly
ascribed to IL-6, our findings revealed a novel IL-6 regulatory program triggered by systemic
thermal therapy that drives a switch to an adhesive vasculature that supports trafficking of
CD8 effector T cells, thereby improving immune-mediated cytolysis of tumor targets. A
concomitant decrease in infiltration of immunosuppressive CD4+ CD25+ FoxP3+ regulatory T
cells (TReg) was further detected following acute systemic thermal therapy that was
15
dependent on IL-6 bioactivity. As a result, we detected a substantial increase in the
intratumoral CD8 TEM:TReg ratio which is considered a strong prognostic indicator of overall
survival in cancer patients. This work suggests that IL-6 derived from non-hematopoietic
stromal cells within the tumor microenvironment can be exploited to create a therapeutic
window that boosts T cell-mediated antitumor immunity. Supported by grants from the
National Institutes of Health (CA79765, CA094045, and AI082039) and the Joanna M.
Nicolay Melanoma Foundation.
Correspondence: [email protected]
16
The Carleton and Sigrid Stewart Keynote Lecture
Saturday, September 29, 2012
11:00 am – 12:00 pm
Room 106B
Flow Cytometry: Let’s Keep this Party Goin’
Jonni Moore, Ph.D.
Professor, Pathology and Laboratory Medicine
Director, Clinical and Research Flow Cytometry
Perelman School of Medicine at the
University of Pennsylvania, Philadelphia, PA
Technologies for single cell analysis have become central to emerging life sciences markets.
Genomics, proteomics and metabolomics are all making daily headlines, but cytomics, or
high dimensional single-cell analyses utilizing systems biology approaches, is rapidly
becoming the technology of choice for the elucidation of relevant phenotypic patterns that
can be directly utilized in the clinical arena. Thus cytometric profiling is poised to play a
major role in research, discovery, diagnosis, prognosis and even therapy. New areas of
application are appearing such as cardiology that will extend the power of flow cytometry to
new, previously untapped areas. While the tools, technology and applications are rapidly
advancing, a significant gap is the lack of experts in the technology. Science careers are not
enticing the new generation so it is up to today’s scientists to encourage the next generation
so that we can “Keep the Party Goin”!
Correspondence: [email protected]
17
Luncheon Roundtables
Rooms 102-105
Saturday, September 29th 12:00-1:30PM
Convener: Paul Champoux, University of Minnesota
Free box lunch available at each table – drinks available at side tables.
The primary aim of these discussions is to provide a forum for participants to address their interests
in a variety of cytometry and image topics.
R1— Future of Flow Cytometry: Moderator: Phil Hexley, Cincinnati Shriner’s Hospital
R2— 10-Parameter Social Media Marketing for Core Management: Moderator: Ryan Duggan,
University of Chicago
R3— Do-it-Yourself Toolkit for Flow Cytometry Repair and Troubleshooting: Moderator: Lisa
Nichols, Cytek Development Inc.
R4— Analysis of Microparticles by Flow Cytoemtry: Moderator: Nancy Fisher, University North
Carolina, Chapel Hill
R5— Personal Cytometers: Bane or Boon to Cores: Moderator: Charles Kucznski, University of
Nebraska
R6— Cell Sorting: Moderator: Justin Meyers, Purdue University
R7— Fluorescent Protein Chemistry: Some New and Some Old but Always Exciting:
Moderator: David LeClerc, University of Chicago
R8— Choosing and Optimizing Funding Opportunities: Moderator: Paul Champoux, University
of Minnesota
R9— Imaging flow cytometry: The Dark Field Rises: Moderator: Hans Minderman, Roswell Park
Cancer Institute
R10— Data Analysis: Moderator: John Quinn, TreeStar, Inc,
Room 102: Tables 1-3 for R1-3
Room 103: Tables 4-6 for R4-6
Room 104: Tables 7-10 for R7-10
Room 105 and Boardroom: for non-participants
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Symposium II
Saturday, September 29th
1:30 pm – 4:15 pm
Emerging Applications of Translational Cytometry
Room 106B
Convener: Michael Sramkoski
All Speakers in this Session are Nakeff Young Investigator Candidates
1:30 – 2:15
Image Cytoemtry-Based Detection of Aneuploidy by FISH-IS.
Orla Maguire, Roswell Park Cancer Institute.
2:15 – 3:00
Characterizing the cross-presentation of cell-associated antigens by
merocytic dentritic cells, a novel DC subset.
Robert I. Thacker, Cincinnati Children’s Hospital.
3:00 – 3:30
Coffee Break (Sponsored by Spherotech, Inc.) – Posters and Exhibits
3:45 – 4:15
Development of flow cytometry techniques for diagnosing and monitoring
the epigenetic status of leukemia patients.
Maria Watson, Prince Margaret Hospital.
Abstracts:
Image Cytometry-Based Detection of Aneuploidy by FISH-IS
Orla Maguire, Ph.D.
Roswell Park Cancer Institute, Buffalo, NY
Fluorescent In Situ Hybridization, (FISH), is a slide-based molecular cytogenetic technique
designed to detect and locate specific nucleic acid sequences. FISH has many applications,
and is one of the conventional methods by which cytogenetic abnormalities are detected in
hematologic malignancies. Sensitivity of this microscopy-based method is limited by the
relatively low numbers of cell events analyzed (usually 200-1000 cells) and the subjective
nature of operator based analysis. We have developed a flow cytometry-based imaging
approach to detect chromosomal abnormalities following FISH in suspension (FISH-IS)
which enables the automated analysis of several log-magnitude higher number of cells
compared to the microscopy-based approach. The present study demonstrates the
applicability of FISH-IS for detecting numerical chromosome aberrations, establishes
accuracy and sensitivity of detection compared to conventional FISH, and feasibility to study
procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor
peripheral blood mononuclear cells hybridized with combinations of chromosome
enumeration probes (CEP) 8, X and Y served as models for disomy, monosomy, and trisomy.
The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was
determined to be 1% with a high level of precision. A high correlation (R2=0.99) with
conventional FISH analysis was found based on the parallel analysis of diagnostic samples
19
procured from 10 AML patients with trisomy 8. Additionally, FISH-IS analysis of samples
procured at the time of clinical remission demonstrated the presence of residual trisomy 8
cells indicating that this approach may be used to detect minimal residual disease and
associated chromosomal defects. The current FISH-IS protocol can be modified for use in
other FISH applications, including diagnosis of syndromes associated with aneuploidy, and
detection of bacteria/pathogens in infection, and for use in other FISH techniques, such as
RNA-FISH.
Correspondence: [email protected]
Characterizing the cross-presentation of cell-associated antigens by
merocytic dendritic cells, a novel DC subset
Robert I. Thacker, Rachel A. Reboulet, Cassandra M. Hennies, Edith M. Janssen
Division of Cellular and Molecular Immunology
Cincinnati Children’s Hospital Research Foundation
University of Cincinnati College of Medicine, Cincinnati, OH
Cross-presentation of cell-associated antigens (Ag) plays an important role in the
induction of anti-tumor responses, autoimmune diseases, and transplant rejection. Recently,
the novel DC subset designated merocytic DCs (mcDCs), has been shown to potently prime
both CD8+ and CD4+ T cells to cell-associated Ag. This priming leads to increased primary
expansion, enhanced effector function, and increased memory formation. The superior
priming of the mcDC is critically dependent on type I IFN production upon uptake of
apoptotic material. Because this breach of tolerance facilitated by mcDCs may prove to be
essential for successful cancer vaccines and interference with autoimmune disease
development, studies to discover distinguishing features among these novel DCs have
become essential.
As the uptake, processing and presentation of cell-associated Ag is highly dynamic
we have utilized multiple tools in our research including ImageStream, flow cytometry,
confocal microscopy and electron microscopy. This multifaceted approach has facilitated the
mechanical distinction of mcDC from other subsets. Comparatively, mcDC take up smaller
particles from apoptotic cells utilizing a Pi3Kinase independent pathway. These small
phagosomes showed a reduced lysosomal acidification rate. In addition, phagocytosed
particles were stored in non-acidic compartments resulting in sustained Ag presentation. The
mcDC are further characterized by type I IFN production, most likely induced by the slow
release of nucleotide structures from early phagosomes wherein apoptotic material is
sequestered. Taken together, these data allow us to correlate functional differences in T-cell
activation to mechanical differences in antigen processing among the DC subsets.
Correspondence: [email protected]
20
Development of flow cytometry techniques for diagnosing and monitoring
the epigenetic status of leukemia patients.
Maria Watson
Prince Margaret Hospital, University of Toronto
Epigenetics is defined as heritable and acquired changes in gene expression or cellular
phenotype caused by mechanisms other than changes in the underlying DNA sequence.
Epigenetic regulation of genes involved in cell growth, survival, or differentiation through
histone modifications have been consistently shown to be an important determinant of cancer
development. There are many post-translational histone modifications but the best
investigated are methylation and acetylation of lysine residues on the histone tails. These
modifications have been shown to influence the structure of chromatin and to affect gene
regulation. For example, H3K9Ac and H3K79me2/3 are associated with transcriptionally
active euchromatin whereas H3K9me2/3 and H3K27me3 are associated with
transcriptionally inactive heterochromatin.
In the current project we are building a multiparametric flow cytometry method for
studying epigenetic regulation via histone modifications. These techniques have been applied
to leukemia patient samples with the goal of developing a robust platform able to link
epigenetic changes to biology and clinical outcome. Once fully optimised we will be able to
use this assay to monitor the effects of drug-like compounds, which target histone-modifying
enzymes, in leukemia cancer patients. We have so far generated novel preliminary data that
establishes the feasibility of building complex single cell assays to monitor histonemodifying enzymes and their specific lysine targets.
There have been several studies into global histone modifications in myeloid
malignancies. Some studies report that depletion of EZH2 along with inhibition of HDACs
leads to apoptosis in AML cells (Fiskus et al, 2009). Others have found inactivating
mutations of EZH2 in various myeloid disorders and the concomitant decrease in
H3K27me3, suggesting EZH2 acts as a tumor suppressor (Ernst et al, 2010). In addition to
this, increased DOT1L methyltransferase activity (a specific methyltransferase of the H3K79
mark) has been linked to MLL-rearranged leukemia (Bernt et al 2011). There have also been
some studies reporting the occurrence of inhibited demethylases in various myeloid
malignancies (Klose et al, 2006, Xu et al, 2011). These studies highlight the importance of
monitoring the epigenetic status in myeloid disorders and the need for an assay to screen for
changes.
Although our clinical focus will be on acute leukemia, where we have access to large
numbers of patient samples, the flow cytometry methods being developed in this project will
be broadly applicable to other types of cancer.
References
Bernt et al, Cancer Cell. 2011 Vol 20(1), 66–78. Ernst et al, Nature genetics. 2010, Vol 42, 722-727. Fiskus et al, Blood. 2009, Vol
114(13), 2733–2743. Klose et al, Nature review, genetics. 2006, Vol 7, 715-727. Xu et al, Cancer cell. 2011, Vol 19, 17-30.
Correspondence: [email protected]
21
Symposium III
Sunday, September 30th
9:00 am – 11:45am
Rare Event Detection
Room 106B
Convener: Amanda (Nicole) White
9:00 - 9:45
From the bench to the bedside: image and flow cytometry analysis of
circulating tumor cells.
Alison L. Allan, London Regional Cnacer Program & Western
University
9:45 – 10:30 Minimal Residual Disease in Acute Myeloid Leukemia. Quatitation by
Flow Cytometry: Why, How, and When?
Anna Porwit, University of Toronto, Universtiy Health Network,
Toronto, ON
10:30 – 11:00 Coffee Break (Sponsored by Cytek Development)
11:00 – 11:45 Cancer Stem Cells: a cell state or a cell type?
Vera Donnenberg, University of Pittsburgh
Abstracts:
From the bench to the bedside:
Image and flow cytometry analysis of circulating tumor cells
Alison L. Allan, Ph.D.
London Regional Cancer Program and Western University, London, ON
Given the multi-step nature of cancer development, there should be several opportunities for
therapeutic targeting of tumor cells and/or the tumor microenvironment. The ideal way to
identify and monitor disease progression is through surrogate marker approaches that are
minimally invasive and allow for longitudinal testing, such as blood tests. Our current
research focuses on the development of such approaches, in particular rare event detection of
circulating tumor cells (CTCs) by image and flow cytometric methods. Identifying rare
populations requires a different approach than standard cytometry techniques, which rely
mainly on positive and negative decisions made in either one, or at most, two dimensional
space. This presentation will discuss the issues that must be addressed when designing an
assay to accurately detect rare populations of CTCs in the blood of patients with solid tumors
and in experimental mouse models of cancer. Cytometry-based methods provide the
opportunity to elucidate the mechanistic details of early steps in cancer progression and
determine how these steps relate to the development, monitoring, and treatment of metastatic
disease.
Correspondence: [email protected]
22
Minimal Residual Disease in Acute Myeloid Leukemia.
Quantitation by Flow Cytometry: Why, How, and When?
Anna Porwit, Ph.D.
Department of Laboratory Medicine and Pathobiology
University of Toronto, University Health Network
Toronto General Hospital, Ontario, Canada
WHY: Minimal residual disease (MRD) monitoring in acute myeloid leukemia (AML) allows
detection of subclinical levels of disease when bone marrow (BM) is morphologically normal. Both
PCR based and multiparameter flow cytometry (FCM) based MRD studies showed that patients with
MRD levels >0.1% of BM cells post induction therapy have higher risk of relapse. Therefore, MRD
levels can be considered as the major independent post-treatment prognostic factor. First preliminary
studies have been published proposing individual pre-transplant assessment, which combined MRD
levels with the well-established pre-treatment prognostic factors such as cytogenetic/molecular
characteristics.
HOW: Sensitivity of MRD detection varies based on the test involved but both PCR and FCM reach
sensitivity 0.01%. For molecular methods multiple probes are required since no single genetic
abnormality is present in more than 50% of patients. FCM detection of MRD is based on abnormal
immunophenotypes displayed by leukemia cells in comparison to normal BM cells. Using modern
FCM, aberrant phenotypes (also called Leukemia Associated Immunophenotypes, LAIP) can be
detected in >90% of patients with AML. For correct interpretation of follow-up samples and detection
of MRD, the immunophenotypic pattern of diagnostic sample and thorough knowledge of
immunophenotype of various cell populations in normal and regenerating BM is necessary. Most
reported studies using 3-5 color FCM included construction of patient specific panels, dependent on
immunophenotype at diagnosis. New 8-10 color approaches (sc. polychromatic FCM) rely on
common comprehensive panels applied at both diagnosis and follow-up, allowing detection of
aberrant cells in most patients using sequential gating strategy.
WHEN: Early time points (day 15 and 30 post-induction) identify as soon as possible high-risk
patients in need of for very intensive treatment. Testing may also help to differentiate leukemic blasts
from regenerating BM in patients with borderline blast numbers in the smears (4-6%). However, there
may be patients with slower blast clearance, which carries some risk for overtreatment. Later time
points (post-consolidation) identify patients with persistent low-level disease, who may be candidates
for SCT. However, the prognostic significance of pre-transplantation MRD levels is still unclear.
CONCLUSIONS: Use of polychromatic MRD assay may increase sensitivity, improve qualitative
information and allow standardization of MRD measurements. Measurement of MRD levels might
refine the prognostic relevance of defined risk-groups based on pre-treatment characteristics.
Correspondence: [email protected]
23
Cancer Stem Cells: a cell state or a cell type?
Vera S. Donnenberg, Albert D. Donnenberg, James B. Hicks, Michael Wigler
University of Pittsburgh, Pittsburgh, PA
Cancer is often viewed as a caricature of normal developmental processes, but the extent to which
its cellular heterogeneity recapitulates the normal multilineage differentiation processes remains
unknown. The classical differentiation scheme, as described in the hematopoietic system, is a
unidirectional differentiation tree with self-replicating stem cells giving rise to progenitor cells
which in turn generate the differentiated progeny.
However not all tissues follow this
unidirectional differentiation paradigm. Experiments from nuclear-somatic cell transfer, from the
creation of induced pluripotent stem cells as well as study of regenerating liver underscore that
stemness may be viewed as a cell state and not a cell type. A change of state resulting in
dedifferentiation of more prevalent “mature” tumor cells into a stem-like tumor phenotype is
compatible with the cancer stem cell paradigm and can only be definitively distinguished from
clonal selection at the single-cell level. Viewing stemness as a state that can be conditionally reexpressed when differentiation signaling pathways are blocked by environment, mutation or
epigenetic reprogramming may help us appreciate the important analogy between tumorigenicity
and normal tissue renewal, without locking us into a one-way differentiation paradigm that views
cancer stem cells as a unique cell type. As the most developed single cell technology, cytometry
and particularly multidimensional cell sorting, provides critical tools for molecular and functional
analysis of cancer cell states. Therefore in this presentation I will review the current state of the art
in the identification and isolation of non-malignant and malignant stem and progenitor cells. I will
address several questions such as: What markers have been used to detect stem cells in normal
human bone marrow, epithelial and adipose tissues? Do markers define populations with
biologically unique properties? Are the same phenotypes found in cancer tissue? Several markers
have been proposed to identify stem/progenitor cells in normal lung and other epithelial tissues. I
will describe a systematic multiparameter investigation of the expression of stem/progenitor
markers on primary clinical tumor isolates in cancer and normal non-malignant tissue. Further I
will demonstrate how the use of immunofluorescence on paraffin sections and multiparameter flow
cytometry determine the histologic location of tumor stem cells. Lastly, I will provide a guidance
concerning avoidance of pitfalls associated with flow cytometry on disaggregated tissues.
Correspondence: [email protected]
24
POSTER ABSTRACTS
Abstract number corresponds to poster board number.
All posters displayed in Rooms 106A&D
1. Quantitative assessment of instrument sensitivity and performance.
Ryan Duggan
UCFlow, The University of Chicago, Chicago, IL
When consumers are looking to purchase a new flow cytometer and are gathering the
specification sheets for instruments from different manufacturers, they typically see a few
common specs; Number and type of lasers, number and type of detectors, max event rate, and
some form of sensitivity measurement - undoubtedly represented by the Detection Threshold
(DT) for FITC and PE. The DT is the number of Molecules of Equivalent Soluble
Fluorophore (MESF) their instrument can detect above electronic noise. Today’s best
instruments typically report somewhere around 100 MESF for FITC and <100 MESF for PE.
With all instruments meeting this ungoverned specification, how is a consumer supposed to
differentiate these instruments? Does having a DT of 50 for FITC make an instrument better
across the board? What about the other 10, or 15 channels on this instrument? Will they
follow suit and perform as well as the FITC and PE channel? Considering that unstained
lymphocytes have MESF values in the FITC channel of approximately 500 units or more, is
the DT even useful as a predictor for instrument sensitivity and resolution? What is
proposed here is a method for instrument sensitivity that can be used by consumers to
yield a composite picture of an instrument’s ability to detect low levels of antigen
expression above autofluorescence background in a quantitative fashion. NORM
(Normalized Overlap Ratio Metric), can be calculated for any fluorochrome that can be
coupled to an antibody, and as such, can allow for the direct comparison of all channels of an
instrument, the same channel between instruments, or the same channel within an instrument
whilst changing filters or laser power, or laser wavelength. The unit of NORM is the
minimum number of antibodies bound to a cell that can be detected above lymphocyte
autofluorescence.
2. Fluorochrome Choice Influences Escapee Formation
Andrew Bantly, Richard Schretzenmair, Lifeng Zhang, Amy Steinmetz, Jonni Moore
Flow Cytometry & Cell Sorting Resource Laboratory
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Introduction:
The apparent loss of mononuclear populations during staining with
fluorescent antibodies, the so-called lymphocyte escapee phenomenon, was noted as early as
1994. The effect was most evident with FITC in ammonium chloride lysed whole blood.
Although there has been a large expansion of fluorescent probes, it has not been determined
if the escapee phenomenon occurs with the new fluorochromes and if it is restricted to certain
subsets. This information is critical to panel design and to accurate measures of individual
leukocyte subsets.
Materials and Methods: Pre-lysed whole blood was stained with CD3 antibodies
conjugated to more than 20 different fluorochromes and analyzed on a BD FACSCanto with
11-color upgrade or a BC Gallios. Events were analyzed using a pan-Leukocyte gate,
excluding only debris and CD3 vs. SSC plots were generated. Escapees were classified as
25
events with CD3 intensity similar to T cells (CD3+ low SSC) which had SSC properties
more appropriate to that of Granulocytes or Monocytes. We also evaluated the use of several
commercial blocking reagents, plasmas and immunoglobulins.
Results: Escapee formation is evident with multiple probes and is dependent upon the
fluorochrome tag and not on the antibody clone. Large difference between donors was noted,
some having virtually no escapees while others with substantial numbers. The use of several
commercial blocking reagents or the addition of murine immunoglobulins had no effect. The
addition of autologous plasma, human AB serum or rabbit sera substantially reduced escapee
formation.
3. Different Sorts for Different Folks: The Importance of Technological Diversity in a
Cell Sorting Facility.
Monica L. DeLay1, A. Nicole White1, Edith Janssen2, George Babcock3, Christopher A.
Worth4, Sherry Thornton1.
1
Research Flow Cytometry Core, Division of Rheumatology and 2Division of Molecular
Immunology, Cincinnati Children’s Hospital, 3333 Burnet Ave., Cincinnati, Ohio 45229,
3
Flow Cytometry Lab, Shriner’s Hospitals for Children-Cincinnati, 3229 Burnet Ave,
Cincinnati, Ohio 45229, 4Stem Cell Institute at James Graham Brown Cancer Center,
University of Louisville, 500 South Floyd Street Louisville, KY 40202
Flow cytometry core facilities have the challenge of offering appropriate technologies to
diverse client needs. Therefore, there is no one-size-fits-all technology that can handle any
and all cell types or applications. For cell sorting, this becomes even more of a challenge
when a variety of cells need to be purified for an even wider variety of downstream
applications. There are two types of technologies currently available for droplet based cell
sorting; cuvette and jet-in-air. While there are advantages and disadvantages to both
technologies, no cell sorter design can fit all needs, especially when the cell type to be sorted
is sensitive to manipulations.
There have been reports (published and anecdotal) of phenotypic and functional changes to
dendritic cells after isolation using different techniques. In our core facility, DC populations
that were sorted on the FACSAria II showed an increase in cell death and were found to be
nonfunctional in an assay testing their ability to process cell associated antigen and stimulate
proliferation of T cells. In contrast, cells sorted on a FACSVantage, MoFlo Legacy or
MoFlo XDP were able to function in the same in vitro assay. We attribute this difference to
the differences in fluid dynamics through the sample path that may damage the cells and
decrease their ability to function. Here we present data comparing the functionality of DCs
with each system. We conclude that it is best to have a diversity of technologies in a cell
sorting facility to better meet the needs of all clients.
26
4. TRAVEL AWARD WINNER - Cell cycle reprogramming blocks apoptosis in
Drosophila follicle cells
Christiane A. Hassel, Brian R. Calvi
Department of Biology, Indiana University, Bloomington, IN
Correct programming of and progression through the canonical cell cycle is necessary to
sustain eukaryotic life. A variation of this cell cycle, the endocycle, is conserved across
species and results in duplication of genomic DNA content without cell division. I have used
the invertebrate Drosophila to explore cell cycle remodeling that results in a mitotic to
endocycle switch (M/E switch) and abrogation of the DNA damage response. When
subjected to irradiation and genotoxic stress, endocycling cells do not apoptose
(Mehrotra et al., 2008). Here, I examine whether experimental induction of an M/E switch
blocks apoptosis.
To induce reprogramming of the mitotic cell cycle, I blocked mitosis by knocking down or
overexpressing mitotic regulators. Specifically, I knocked down cyclin A (cycA) or
overexpressed fizzy-related (fzr/cdh1). cycA RNAi or fzr/cdh1 overexpression resulted in an
M/E switch in ovary follicle cells. When subjected to gamma-irradiation, apoptosis was
repressed in these cells. This shows that an endocycle can be induced by alteration of a single
mitotic regulator and this is sufficient to repress apoptosis.
These data suggest that cell cycle reprogramming in some cells is sufficient to repress
apoptosis. Some tumors may have chemotherapy-resistant endocycling cells. Further
investigating how cell cycle reprogramming is linked to apoptosis-resistance may lead to the
discovery of new cancer treatments.
5. Modification of a Canto A for Simultaneous 10 Fluorescent Color Detection.
Edward Podniesinski, Alexis Conway, Terry Donahue, Aileen Cinquino, Paul Wallace
Laboratory of Flow & Image Cytometry, Roswell Park Cancer Institute, Buffalo N.Y. 14263
The number of fluorochromes conjugated to monoclonal antibodies commercially available
for routine flow cytometry continues to grow. With the addition of PE Texas Red/PE-CF594
and Alexa 700 flours routine 10 color flow cytometry is now a reality. Our original 6 color
Canto-A’s instrument architecture (4/2) was first upgraded to 3 lasers within the recent past
through a purchase of a SORP (Special Order Research Option). This produced a
fluorescence emission detection count on par with the CANTO-II (4/2/2). We found the
opportunity existed to easily add two additional spectral detection channels to the existing 3
laser interrogation/ intercepts.
To increase the current 3 laser CANTO-A detection capability to 10 color (5/3/2), required 2
additional PMTs (Photo Multiplier Tubes), 2 integrated PMT/ HV control sockets, 2 remote
Pre-Amplifier circuit boards, Red/PE-CF594 filter/ mirror set, Alexa 700 filter/ mirror set
and some additional cabling. The CANTO-A software configuration needed change
necessary to map or associate the 2 additional detectors for acquisition.
The performance characteristics of the Canto A was compared to itself before and after
upgrading. We will show our findings as a result of the upgrade from 8 to 10 color.
27
6. Performance Evaluation of A Flow Cytometer’s Optical Filters From New to 8 years
Later
Edward Podniesinski, Paul Wallace
Roswell Park Cancer Institute, Buffalo, NY
The detection performance of aged optical filters can get overlooked from Flow Cytometers
of long service. An 8 year old flow cytometer’s detection optics were spectrally swept by a
slightly modified spectrophotometer and compared to when the filters were new and to new
identical replacements. Data from the spectrometer and multi-peak broad spectrum
calibration particles, used to critique detector channel performance, will also be presented.
7. Assessing Containment and De-Contamination of Cell Sorters
Matthew Cochran
University of Rochester, Rochester, NY
Contamination of aseptic sorts can cause frustration on the part of the investigator, not to
mention the lost time and effort caused by this problem. Many sorting facilities take special
care to prepare and maintain their instruments in a ‘clean’ state using different
decontamination procedures to minimize the chance that the system is the source of
contamination. In multi-user facilities where samples range from mammalian primary cells
and cell lines to yeast and bacteria, cleaning protocols between sorts are especially important.
Coupled to this is the fact that many testing protocols require 24-48 hours after a sort to
determine if the system was clean, which may be too late for the investigators who are
possibly impacted by the contamination. Our current protocols use a permissive plate to
detect aerobic bacteria at concentrations as low as 25/ml, but take 48 hours to complete.
Recently BD Biosciences released a flow-based assay for detecting bacterial contamination,
the FACSMicro count. The system is composed of a flow cytometer and bacterial testing
reagents, offering results in as little as 5 minutes, with sensitivities between 30-50
bacteria/ml. In this report, we discuss the optimization of these reagents for use with
standard flow cytometers and compare it to current plate based testing protocols used in our
facility.
8. Nuclear translocation of NFAT and NF-κB in T-cells as an early quantitative
response parameter of immune-activity pre- and post-solid organ transplantation.
Orla Maguire, Kieran O’Loughlin, Oleh Pankewycz, Mark R. Laftavi, Hans Minderman
Roswell Park Cancer Institute, Buffalo NY
With the development of more effective immunosuppressive therapies as well as better
combination strategies for their use, the rates of acute rejection after kidney transplantation
have plummeted to single digit levels and one year graft survival rates of 90%. Paradoxically,
this early success has not led to improved long-term allograft survival. Durable graft survival
over the course of years depends on finding the right balance between overimmunosuppression leading to drug toxicity or infectious complications and underimmunosuppression resulting in the formation of donor specific antibodies and chronic
rejection both of which cause premature graft failure. Effective post-transplant patient
management lacks an adequate method for determining the optimal level of
immunosuppression required for a given individual to prevent toxicity or rejection. We aim
28
to develop an effective early-response assay which can gauge the overall immune status in
the post-transplant recipient, thus, allowing for personalized therapy. The most commonly
used immunosuppressants function by inhibiting the activation of the transcription factors
NFAT and NF-κB, thereby reducing the transcription of immune response genes. The
function of these transcription factors depend on their translocation from the cytoplasm to the
nucleus which is quantifiable by imaging cytometry. Preliminary studies in our lab show that
we can measure transcription factor activation in T-cells using HLA (Human Leukocyte
Antigen) coated beads which corresponds to a proliferative response. A peak in signaling
activity is demonstrated at 16hrs post-stimulation. Proliferation is measurable 7 days
following stimulation. This time frame is longer than e.g., CD3/CD28 stimulation, where
signaling occurs within a few hours, and proliferation can be measured in four days. We
believe this difference is due to the increased specificity of the anti-HLA response. Using the
immunosuppressant tacrolimus, we can inhibit CD3/CD28-induced NFAT and NF-κB
nuclear localization in healthy donor PBMCs ex vivo at concentrations equivalent to those
achieved in post-transplant patients. These early results indicate that this assay could be
useful in measuring potential reactivity towards the donor graft before/immediately following
transplant and monitoring immunosuppressant activity in patients in the months following
transplant.
9. Sorting Renin Positive Cells from Mouse Bone Marrow
Craig A Jones, Joseph Tario, Paul Wallace and Kenneth W. Gross
Roswell Park Cancer Institute, Buffalo, NY
The existence of a functional Renin-Angiotensin system in mammalian bone marrow has
been postulated for many years. In fact, expression of many of the genes comprising the RAS
has been documented. However, evidence of renin expression, which catalyzes the first step
of this enzymatic cascade, in normal bone marrow has been sketchy. We use PCR and RTPCR to confirm expression of the renin gene in mouse bone marrow cells. Furthermore,
Affymetrix microarrays were used to characterize renin-expressing cells sorted from the bone
marrow of transgenic mice carrying a renin-GFP reporter gene. In addition, immunephenotyping was used to show these cells were multipotent and had characteristics of early
B-cell lineage.
10. Statistical classification of multivariate flow cytometry data analyzed by manual
gating: Stem, progenitor and epithelial marker expression in non-small cell lung cancer
and normal lung
Bratislav Janjic, Albert D. Donnenberg, Vera S. Donnenberg, Daniel P. Normolle
UPMC, University of Pittsburgh, Pittsburgh, PA
The use of unsupervised classification to extract markers from primary flow cytometry data
is an emerging field which has made significant progress, spurred by the growing complexity
of multidimensional flow cytometry. Whether markers are extracted without supervision or
by conventional gate and region methods, the number of candidate variables identified is
typically larger than the number of specimens (p > n) and many are highly inter-correlated.
Thus comparison across groups or treatments to determine which markers are significant is
challenging. Here we utilize a data set in which 86 variables are created by conventional
manual analysis of individual listmode data files, and compare the application of five
multivariate classification methods to discern subtle differences between the stem/progenitor
29
content of 35 non-small cell lung cancer and adjacent normal lung specimens. The methods
compared included elastic-net, lasso, random forest, diagonal linear discriminant analysis and
best single variable (best-1). We describe a broadly applicable methodology consisting of: 1)
Variable transformation and standardization; 2) Visualization and assessment of correlation
between variables; 3) Selection of significant variables and modeling; and 4)
Characterization of the quality and stability of the model. The analysis yields both validating
results (tumors are aneuploid and have higher light scatter properties than normal lung), as
well as clues that require followup: Cytokeratin+ CD133+ progenitors are present in normal
lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more
prevalent in tumor. We anticipate that the methods described here will be broadly applicable
to a variety of multidimensional cytometry problems.
11. Perforin Deficienc Presenting in an Adolescent as IBD
Trivikram Dasu1, Jerome Sigua2, Cindy Bauer2, James Casper3, John Routes2
and James Verbsky1,4
1
Clinical Immunodiagnostic & Research Laboratory, Medical College of Wisconsin
2
Division of Pediatric Allergy, Asthma & Clinical Immunology
3
Divisions of Pediatric Hematology-Oncology, Blood & Marrow Transplantation
4
Division of Pediatric Rheumatology, Children’s Hospital of Wisconsin, Milwaukee, WI
A 14 year-old Caucasian male with a 1-year history of ulcerative colitis presented with
increased ostomy output, vomiting, abdominal pain, anorexia, and fatigue 1-month postcolectomy. Endoscopy showed new onset pan-enteritis.
Tests for humoral deficiency, Chronic Granulomatous Disease, Toll-like receptor 4, Immune
Dysregulation Polyendocrinopathy Enteropathy X-linked, X-linked Lymphoproliferative
Disease, and NFkB Essential Modulator Deficiency were normal. Natural Killer (NK) cell
cytotoxicity was severely reduced with normal absolute NK cell numbers.
Immunophenotyping for expression of perforin and granzymes revealed perforin deficiency
in cytotoxic NK and CD8 T cells. Genetic testing demonstrated a homozygous PRF1 272
C>T (A91V) mutation, which has been reported to be a disease causing variant in familial
hemophagocytic lymphohistiocytosis (HLH). Furthermore, the patient’s lymphokine
activated NK cells did not upregulate expression of perforin in presence of IL-2 compared to
the control cells.
The patient’s fraternal twin brother demonstrated detectable, but reduced perforin expression
in his lymphocytes, indicating haploinsufficiency. The subject’s sister and mother, however,
displayed normal expression levels and profiles of perforin and granzymes in the cytotoxic
lymphocyte subsets.
Rituximab and immunoglobulin replacement were initiated while awaiting evaluation for
allogenic bone marrow transplant. This report demonstrates that perforin deficiency, one of
the familial causes of HLH, can also present as inflammatory bowel disease in adolescence.
30
12. Hyperbaric oxygen stimulates adipose- derived mesenchymal stem cells growth and
differentiation in streptozocin-induced diabetes mellitus type 1 mice in vivo
Tatyana N. Milovanova1, Veena M. Bhopale1, Sorokina Elena M1 ,Gunalp Uzun1,
Ming Yang1, Jonni S. Moore3 and Stephen R. Thom 1,2
1
Institute for Environmental Medicine, 2Departments of Pathology and Laboratory Medicine
and Emergency Medicine, University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania; 3Department of Flow Cytometry, University of Pennsylvania Medical Center,
Philadelphia.
Hyperbaric oxygen modified function of stem/progenitor cell (SPCs) in new blood vessels
formation which is a required step in wound healing in diabetic patients. In wounds healing
VEGF secreted from adipose-derived mesenchymal stem cells (ASCs) induces migration and
proliferation of endothelial cells, increasing the vascularity of wound bed. We hypothesized
that oxidative stress from hyperbaric oxygen (HBO2, 2.8 ATA for 90 min daily) exerts a
trophic effect on adipose- derived mesenchymal stem cells in streptozocin-induced diabetes
mellitus type 1 mouse model via reactive oxygen /reactive nitrogen species (ROS/RNS)dependent mechanism. Adipose-derived stem/progenitor cells (ASCs:Sca-1+/CD31-/DAPI-),
were sorted from omentum of additional WT mouse, labeled with the fluorescent dye 5,6carboxyfluorescein diacetate succinimidyl ester (CFSE), added to one of the Matrigel plugs
subcutaneously injected one on either side of the thoracic vertebrae and stimulated by HBO2.
In combination, HBO2 and ASCs in vivo demonstrated cumulative effects. Vascular channels
lined by CD34+ SPCs were identified and they were significantly reduced in diabetic
animals. In the Matrigel with seeded ASCs after HBO2 simulation accelerated channel
development, cell differentiation based on surface marker expression and cell cycle entry was
identified. CD34+ SPCs of diabetic animals in blood and bone marrow defined down
regulated thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3,
phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and
stromal cell-derived factor-1 in. Cell recruitment to Matrigel and protein synthesis responses
was abrogated in STZ-mice. Thioredoxin system activation leads to elevations in HIF-1 and 2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on
SPCs. By causing an oxidative stress, HBO2 activates a physiological redox-active autocrine
loop in recruited SPCs and increased paracrine secretion in seeded ASCs, resulted in the
stimulated neovasculogenesis.
13. TRAVEL AWARD WINNER - Looking at Microvesicles (MVs) by Flow Cytometry
Phil Hexley
Shriners Hospitals for Children, Cincinnati, OH
Microvesicles (MVs) are small blebs released from mammalian cells following apoptosis or
activation. With mounting evidence suggesting MVs act as distal effectors of pathology, they
are increasingly relevant in clinical settings. However, due to their size range (0.1 – 1 µm
diameter) and relative novelty, they are notoriously difficult to measure: even with known
caveats of flow cytometry in this size range it is still a popular method of analysis. In
addition, many analytical and patient variables have been shown to artificially increase
circulating plasma MV numbers yet protocol standardization is still lacking. We
acknowledge there are still limitations, but by controlling and/or minimizing known
31
confounding variables, we obtained data indicating detectable circulating plasma MV
number correlates strongly with total body weight (R2=0.91). The relationship we have
observed should be an important point to consider in future research into circulating plasma
MVs, furthermore the consistency in intra-donor sampling over time may be encouraging for
using this number as a clinical parameter.
14. Cross Platform Solutions for Autophagy Detection using the guava 8HT and Amnis
ImageStream mkII
Mark Santos, Kevin Su, Haley R. Pugsley, Angelica Olcott, Jason Whalley, David Basiji,
Patrick Schneider, and Matthew Hsu
EMD Millipore, Hayward, CA, United States
Autophagy is an intracellular catabolic pathway which causes cellular protein and organelle
turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and
Crohn's disease, in addition to aging. It is a tightly regulated process that plays a normal part
in cell growth, development, and cellular homeostasis. Autophagy functions as a
housekeeping mechanism through disposal of aging and dysfunctional proteins and
organelles by sequestering and priming them for lysosomal degradation Increasing evidence
suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly
influence general cell health. Malfunctions of autophagy can adversely impact longevity and
the capability of cells to function at full capacity.
Here, we describe two key platforms paired with a novel assay design which will allow for
the study of autophagy by flow cytometry. Guava 8HT systems have the capabilities of
higher throughput sampling utilizing a 96-well format and smaller sample sizes for
acquisition. And when combined with the InCyte software module for data analysis and
curve fitting algorithms for small molecule analysis, one can now screen autophagy inducers
or inhibitors more efficiently. Amnis’s ImageStream system is a perfect complementary
platform as it combines the capabilities of microscopy and flow cytometry in a single
platform for quantitative image based cellular analysis of autophagy.
15. Enhanced Dynamic Range Capabilities for Microcapillary Cytometry
Katherine Gillis, Asima Khan, Julie Clor, Kamala Tyagarajan, Ray Lefebvre, Bruce
Goldman, Rick Pittaro
EMD Millipore, Hayward, CA, United States
Sensitive and multi-parametric cell analysis by flow cytometry typically requires substantial
expertise, a confusing array of protocols, and cumbersome software analysis, thereby limiting
its utility for on-demand use. Flow cytometric set-up can often be cumbersome and time
consuming as it requires multiple steps for instrument configuration and data analysis.
Samples often require gain adjustment to view positive and negative samples on screen, or
else both populations cannot be visualized together. Conversely, small shifts within
experiments may be overlooked if display scaling is not utilized to its fullest. In this work,
we demonstrate how features of InCyteTM software allow the user to not only display samples
comparing large fluorescence shifts and minimal fluorescence shifts, such as GFP expressing
cells and CFSE stained cells, but additionally we highlight how small fluorescent shifts in
32
Cytochrome c loss from the mitochondria can be detected with no loss in data integrity.
Additionally, utilizing features such as six-parameter heat-mapping and IC50/EC50 curve
generation offer sophisticated data analysis in just a few steps, with updates in real time and
easy export of data. As a result, users now have complete flexibility regarding their choice of
assay and analysis methods. Experiments can utilize a set of samples run in a single day,
across multiple days, or even between multiple assays or experimental questions. Taken as a
whole, these novel and innovative advancements of InCyteTM Software enable analysis of
multiparametric cellular data in a format which is even faster, simpler and more accurate.
16. Simplified Cell Cycle Analysis on the MuseTM Cell Analyzer
Katherine Gillis, Asima Khan, Kimvan Tran, Arni Barican, Anh Nguyen, Ray Lefebvre,
Kamala Tyagarajan
EMD Millipore, Hayward, CA, United States
Cell Cycle analysis and the assessment of cell cycle impacts has traditionally been restricted
to more expensive and elaborate instrumentation platforms that required greater user
expertise. In this study we present highly simplified cell cycle analysis solutions on a novel,
cytometry platform, the Muse Cell Analyzer. The Muse Cell Analyzer is a small footprint
instrument that utilizes innovative optics and electronics along with a touch-screen interface
to guide users to obtain quantitative cellular data and obtain comprehensive cell health
analysis.
In this study, we present results using the Muse Cell Cycle Assay and dedicated software
module, for the identification of cells in different phases of the cell cycle. Using suspension
(Jurkat) and adherent (PC3) cell line changes in the phases of the cell cycle could be clearly
distinguished using compounds that are know to arrest cell cycle phases, nocodazole and
aphidicolin as well as compounds that are known apoptotic inducers, staurosporine and
etoposide. Impacts on both cell count and apoptotic percentages were evaluated in parallel
using other count and viability and Annexin-V based assays on the platform. Comparative
studies with predicate methods established that comparable and accurate results for
determination of % of cells in different phases of cell cycle could be obtained on the novel
system. The simplicity of the assay and data interface and its output further allowed for
highly precise results to be obtained on the platform with %CV of replicates being <10%.
The Muse Cell Cycle Assay solution thus greatly simplifies a traditionally complex assay and
makes it accessible to users of multiple expertise level while still offering rapid, accurate and
precise cell cycle results.
17. Approaches to Evaluate Immune Cell Health and Apoptosis Using MicroCapillary
Cytometry
Julie Clor, Kimvan Tran, Katherine Gillis and Kamala Tyagarajan
EMD Millipore Corporation, Hayward, CA
The study of apoptosis and cell death mechanisms in immune cell subpopulations has
become increasingly important to understand the pathways that lead to elimination of cells
during normal development, disease, drug treatment or autoimmune responses. These studies
are complicated and cumbersome to perform since they require parallel identification of
immune cells and identification of impact of the treatment condition on immune cell health
33
and often have to be performed on limited sample sizes. We present here highly simplified
approaches using microcapillary cytometry and small cell sample sizes to investigate changes
in annexin V–based apoptosis detection, mitochondrial membrane potential changes, FAS
expression levels and caspase activity in in CD4 and CD8 T cell populations simultaneously
using a series of FlowCellect Immune Cell Health kits. The approaches thus provide
information in change in immune cell population and a snapshot of immune cell health. Our
results demonstrate that the approaches can be applied to plate-based screening studies to
screen PBMC’s with ~80 cytotoxic compounds from blood donors. The studies allowed for
the rapid identification of compounds such as thimerosal, phenylmercuric acetate, 2,6
dimethoxyquinone and sanguinarine sulfate that caused high levels of apoptosis in immune
sub-populations and provide mechanistic information on their mode of action. The facile
identification and dissection of immune cell apoptosis pathways can greatly enhance our
understanding of how compounds interact with the immune system.
In a screening study with 80 cytotoxic compounds, PBMC’s from multiple donors were
treated with cytotoxic compounds and then evaluated for their impacts on CD4 and CD8 T
cells and their immune cell health using immune cell health assays followed by flow
cytometry on the guava easyCyte cytometry platform. The studies identified several
compounds such as gambogic acid, thimerosal, phenylmercuric acetate, 2,6
dimethoxyquinone and sanguinarine sulfate which caused high levels of apoptosis in both
CD4 and CD8 T cells with overnight treatment with some of them having differential effects
on individual sub-populations.
18. A Networking Guide for A Multi-parametric Community
White, AN; Delay, ML; Thornton, S
Cincinnati Children’s Hospital Medical Center
Networking is the framework essential to building relationships, forming collaborations, and
the exchanging ideas. The field of cytometry is a multi-faceted community of researchers,
suppliers, and technical specialists from varying areas of disciplinary interests and expertise.
These indviduals come from both academic and industrial backgrounds and are trained in
different disciplinary fields of study in engineering, biology and chemistry. The
diversification, along with geographical location and access to funding for travel to meetings
can also inhibit the exchange of ideas between heterogenous groups. The complex
networking structure in a field like cytometry introduces several potential impediments that
work against a network’s competency to achieve productive mechanisms for the exchange of
ideas. Utilizing established organizations and affiliations within the cytometry community
can (1)enhance its networking competency between local, regional, and international groups;
(2) create a stronger interconnectedness among diverse cytometrists; and (3) increase the
degree of impact cytometrists have on each other.
34
19. Analysis and Sorting of Fragile Cells (Adipocytes, Protoplasts etc.), Cell Clusters
and Model Organisms Using BIOSORTER® Instrument
Mikalai Malinouski, PhD,
Union Biometrica, Inc.
Many objects are too large or fragile for conventional flow cytometry. Union Biometrica
instruments are engineered to automate the analysis and sorting of fragile cells and “large”
objects such as hepatocytes, adipocytes, cardiomyocytes, kidney tubular cells, plant
protoplasts, embryoid bodies, pancreatic islets. Other suitable samples are small model
organisms such as all stages of C.elegans, Drosophila eggs, embryos and larvae, and
Zebrafish eggs, embryos and the early larval stages. The BioSorter instrument uses
interchangeable fluidic modules and is capable of analyzing and sorting a wide range (11,500 micron) of object sizes at rates of 10-500 objects/second. Using object size, optical
density and fluorescence intensity as sorting criteria, selected objects can be dispensed in
multi-well plates or bulk receptacles. A gentle pneumatic sorting mechanism avoids harming
sensitive objects thereby making the instrument suitable for fragile cells and live biological
materials.
Several applications (e.g. model organisms) require extensive analysis of object shape and
fluorescence localization before sorting. A unique feature available with these instruments is
Profiler II™ which allows setting complex sorting parameters. Each object passing through
the flow cell is digitized into a 2D profile. These axial profiles are analyzed and can be used
as sort criteria based on the localization of optical density and fluorescence within each
object, including peak height, peak width, peak counting and relative peak position. This
approach is much faster than imaging and proves to be useful in the analysis and sorting of
cells, cell clusters and model organisms.
35
GLIIFCA STEERING COMMITTEE - 2012
Contact info
Canada
David Hedley
California
Maurice R. G. O’Gorman
Illinois
Ryan Duggan
Indiana
Lisa Green
Bartek Rajwa
Iowa
Bruce Pesch
Massachusetts
Betsy Ohlsson-Wilhelm
Michigan
Alexander Nakeff
Louis King
Minnesota
Paul Champoux
New York
Matt Cochran
Sally Quataert
Paul Wallace
Ohio
R. Michael Sramkoski
A. Nicole White
Pennsylvania
Vera Donnenberg
Mike Meyer
Andrew Bantly
Tennessee
Keith Shults
Washington DC
Karen Domenico
Wisconsin
Katharine Muirhead
Brian DuChateau
Kathy Schell
Emeritus
Carleton & Sigrid Stewart
Charles Goolsby
[email protected]
2012 President
[email protected]
[email protected]
Co-Executive Secretary
[email protected]
[email protected]
Education & CMLE Chair
[email protected]
[email protected]
[email protected]
[email protected]
Site Committee Chair
[email protected]
Roundtable Chair
[email protected]
[email protected]
[email protected]
Core Manager Meeting Chair
Treasurer
[email protected]
[email protected]
2012 Program Committee
2012 Program Committee
[email protected]
[email protected]
[email protected]
Co-Executive Secretary
[email protected]
[email protected]
Vendor Liaison
[email protected]
[email protected]
[email protected]
Treasurer
[email protected]
[email protected]
Emeritus Members
Emeritus Member
36
GLIIFCA 21 Registered Attendees
First Name
Last Name
Email address
Institution
David
Adams
[email protected]
University of Michigan
Alison
Allan
[email protected]
London Regional Cancer Program
David
Allman
[email protected]
University of Pennsylvania
George
Babcock
[email protected]
Shriners Hospital for Children
Matthew
Balys
[email protected]
University of Rochester
Andrew
Bantly
[email protected]
University of Pennsylvania
Suzanne
Buck
[email protected]
Life Technologies
Tim
Bushnell
[email protected]
University of Rochester
Paul
Champoux
[email protected]
University of Minnesota
Juan
Chen
[email protected]
University of Illinois at Chicago
Carolyn
Cheney
[email protected]
Ohio State University
Sue
Chow
[email protected]
Princess Margaret Hospital
Aileen
Cinquino
[email protected]
Roswell Park Cancer Institute
Matthew
Cochran
[email protected]
University of Rochester
Bunny Anne
Cotleur
[email protected]
Cleveland Clinic
Kamila
Czechowska-Kusio
[email protected]
Yale Medical School
Trivikram
Dasu
[email protected]
Medical College of Wisconsin
Monica
DeLay
[email protected]
Cincinnati Children's Hospital
Priscilla
Do
[email protected]
The Ohio State University
Ree
Dolnick
[email protected]
Roswell Park Cancer Institute
Karen
Domenico
[email protected]
Children’s National Medical Center
Terry
Donahue
[email protected]
Roswell Park Cancer Institute
Vera
Donnenberg
[email protected]
University of Pittsburgh
Albert
Donnenberg
[email protected]
University of Pittsburgh
Ryan
Duggan
[email protected]
University of Chicago
Ann Marie
Eckel
[email protected]
Roswell Park Cancer Institute
Masilamani
Elangovan
[email protected]
Idea Elan
Karen
Ersland
[email protected]
UWCCC Flow Cytometry Laboratory
Sharon
Evans
[email protected]
Roswell Park Cancer Institute
Nancy
Fisher
[email protected]
University of North Carolina
Rosemary
Furlage
[email protected]
Roswell Park Cancer Institute
Balaji
Ganesh
[email protected]
University of Illinois at Chicago
Jeannie
Gaylor
[email protected]
DVS Sciences
Jewell
Graves
[email protected]
University of Illinois at Chicago
Christiane
Hassel
[email protected]
Indiana University
David
Hedley
[email protected]
Princess Margaret Hospital
Erika
Heninger
[email protected]
UWCCC Flow Cytometry Laboratory
Phil
Hexley
[email protected]
Shriners Hospital for Children
Jan
Hoffman
[email protected]
Roswell Park Cancer Institute
Chris
Holley
[email protected]
Washington University Siteman CC
Matthem
Hsu
[email protected]
EMD Millipore
Jackie
Hughes
[email protected]
Washington University Siteman CC
Bratislav
Janic
[email protected]
Pittsburgh Cancer Institute, U. of Pitt.
James
Jacobberger
[email protected]
Case Western Reserve University
Craig
Jones
[email protected]
Roswell Park Cancer Institute
Louis
King
[email protected]
Michigan State University
Geoff
Kraker
[email protected]
Northwestern University
Charles
Kuszynski
[email protected]
University of NE Medical Center
Joanne
Lannigan
[email protected]
University of Virginia
37
David
Leclerc
[email protected]
University of Chicago
Deborah
Lee
[email protected]
Bristol-Myers Squibb
Orla
Maguire
[email protected]
Roswell Park Cancer Institute
Roland
Marcus
[email protected]
FluoroFinder
Bryan
Mc Elwain
[email protected]
The Ohio State University
Mike
Meyer
[email protected]
University of Pittsburgh
Justin
Meyers
[email protected]
Purdue University
Tatyana
Milovanova
[email protected]
University of Pennsylvania, IFEM
Hans
Minderman
[email protected]
Roswell Park Cancer Institute
Jonni
Moore
[email protected]
University of Pennsylvania
Alex
Nakeff
[email protected]
Carole Anne
Nakeff
[email protected]
Jeffrey
Nelson
[email protected]
Northwestern University
Mario
Nunez
[email protected]
Beckman Coulter
Kieran
O’loughlin
kieran.o’[email protected]
Roswell Park Cancer Institute
Betsy
Ohlsson-Wilhelm
[email protected]
SciGro, Inc./NorthEast Office
Mike
Olson
[email protected]
University of Chicago
Andrew
Osterburg
[email protected]
University of Cincinnati
Jennifer
Piraino
[email protected]
Roswell Park Cancer Institute
Hank
Pletcher
[email protected]
University of Pennsylvania
Edward
Podniesinski
[email protected]
Roswell Park Cancer Institute
Anna
Porwit
[email protected]
Toronto General Hospital
Sally
Quataert
[email protected]
University of Rochester
Aaron
Robida
[email protected]
University of Michigan
Wade
Rogers
[email protected]
University of Pennsylvania
Kathleen
Schell
[email protected]
UW Retired
Heather
Schumacher
[email protected]
Van Andel Research Institute
Dan
Schweppe
[email protected]
Washington University Siteman CC
Dagna
Sheerar
[email protected]
UWCCC Flow Cytometry Laboratory
Keith
Shults
[email protected]
IncellDX,Inc.
Sukhwinder
Singh
[email protected]
UMDNJ – New Jersey Medical
Sharleen
Slaunwhite
[email protected]
University of Rochester
Michael
Sramkoski
[email protected]
Case Western Reserve Univ
Kim
Stojka
[email protected]
Joseph
Tario, Jr.
[email protected]
Roswell Park Cancer Institute
Robert
Thacker
[email protected]
Cincinnati Childrens Hospital
Sherry
Thorton
[email protected]
Cincinnati Children’s Research
Rachel
VanWinkle
[email protected]
Beckman Coulter
Paul
Wallace
[email protected]
Roswell Park Cancer Institute
Maria
Watson
[email protected]
Prince Margaret Hospital
Nicole
White
[email protected]
Cincinnati Children’s Hospital
Wojciech
Wojciechowski
[email protected]
University of Rochester
Philip
Woost
[email protected]
Case Western Reserve University
Daniel
Wu
[email protected]
Princess Margaret Hospital
38
Registered Vendor Representatives
Alexion Pharmaceuticals, Cheshire, CT
http://www.alxn.com/
Dan Hayden
Kim Coleman Lohr
DVS Sciences, Inc., Sunnydale, CA
http://www.dvssciences.com/
Jeannia Gaylor
Clare Rogers
Amnis Corporation (see EMD Millipore)
eBioscience, San Diego, CA
www.ebioscience.com
Elizabeth Dobbs
Hether Ide
Keith Olszens
Susan Reynolds
Bay Bioscience, San Francisco, CA
www.baybio.co.jp
David Coder
Beckman Coulter, Miami, FL
www.beckmancoulter.com
Cheryl Guyre
Peter Racanelli
Patricia Sardina
John Stojka
EMD Millipore,
http://www.millipore.com/
Andy Arena
Darin Fogg
Sabrina Hawthorne
Dave Kennedy
Cassandra Singer
Christine Valway
BD Biosciences, San Jose, CA
www.bdbiosciences.com
Eva Chan
Darci Gorgone
Bill Gunderman
Janet Horta
Thomas Lendrihas
Susan Merill
Nicole Ovadia
iCyt Mission Technology, Champaign, IL
www.i-cyt.com
Matt Alexander
Joanie Capps
Jeff Clapper
Mike Kanavos
Sharlene Wright
BioLegend, San Diego, CA
www.biolegend.com
Rebecca Bultema
Thomas Williams
Immudex, Copenhagen, Denmark
http://www.immudex.com/
Stephen Haley
Kivin Jakobsen
Cedarlane Labs,
http://www.cedarlanelabs.com/
Joel Koolstra
Leinco Technologies, St. Louis, MO
http://www.leinco.com/
Patrick Leinert
Cell Signaling Technology, Beverly, MA
www.cellsignal.com
Gordon Miller
Life Technologies, Eugene, OR
http://www.lifetechnologies.com/home.html
Jolene Bradford
Neil Bristol
Suzanne Buck
Dolores Ciufo
Pat Jacobs
Barb Sereick
Cytek Development, Gordonsville, VA
www.cytek.com
Kim Belenzom
Raymond Lannigan
Lisa Nichols
Nick O’Gorman
Miltenyi Biotec, Inc., Auburn, CA
www.miltenyibiotec.com
Dan Graziano
Shane Oram
Ashley Weant
De Novo Software, Thornhill, Ontario
www.denovosoftware.com
David Novo
39
Partec North America, Inc., Swedesboro, NJ
www.partec.com
Roy Overton
Allan Schell
Stratedigm, Inc., San Jose, CA
http://stratedigm.com/
Thermo Fisher (Dako), Waltham, MA
http://www.thermofisher.com/global/en/home.asp
Brian DuChateau
Mike Stinson
Propel Labs, Fort Collins, CO
http://www.propel-labs.com/
George McDonough
TreeStar, Inc., Ashland, OR
www.treestar.com
John Quinn
Nick Ostrout
R&D Systems, Minneapolis, MN
http://www.rndsystems.com
Chris Larson
Union Biometrica, Holliston MA
http://www.unionbio.com/
Michael Fazzio
Mikalai Malinouski
Spherotech, Inc, Libertyville, IL
www.spherotech.com
Andy Wang
Brian Shah
Ayesha Hamid
Verity Software House, Topsham, ME
www.vsh.com
Mark Munson
40
NOTES:
41