How to define flow cytometry ? Méthodes d’étude de la cellule
How to define flow cytometry ? Méthodes d’étude de la cellule The possibility to measure individually and simultaneously several MV426 physical and biological characteristics of a cell in a heterogeneous solution. It allows the identification of subpopulations and the Flow cytometry… estimation of an average population To measure (-metry) optical cells properties (cyto-) through a fluid High rate (5.104 cells/s) and high sensitivity (100 antigenic (flow) in front of an excitatory laser beam… factors/cell) The possibility of separating physically every cell analysed Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 CyFlow® Cube - Partec LSR Fortessa Becton Dickinson Historic 1934 Moldavan - Cellular numeration : capillary + photoelectrique captor 1949 Coulter - Count, size (conductance variability) 1953 Crosland -Taylor - Use laminar flow as describes by Reynolds in 1883 1965 Kamentsky - Analysis of cells constituents (2 parameters : DNA + Proteins) Fulwyler - Cell sorting using electrostatic methods Gallios Beckman Coulter Some examples of… 1969 Van Dilla - Laser excitation Flow cytometers : analysis 1972 Herzenberg - Cells sorting (1st article et 1st cytometers commercialization) 1978 Schlossman - Monoclonal antibodies production Reinhertz - New fluorochromes development 2004 Perfetto - Analyze of 19 parameters (17 fluorescent signals) 2006 Chattopadhyay - Immunophenotypage by quantum Dots Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 EasyCyte8HT Guava Technologies MACSQuant MiltenyiBiotec MoFlo XDP Beckman Coulter C6 Accuri Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Flow cytometry principle 1) Fluidic system Astrios Beckman Coulter Some examples of… 3) Electronic system Presents Particles in front of the laser beam Allows the conversion from the different luminous signals to electronic signals which can be stored and processed by the computer via an appropriate software Flow cytometers: analysis and cell sorting Influx Becton Dickinson FACSAria Becton Dickinson 2) Optical system Invitrogen Composed of one (or more) monochromatic excitation source(s) (LASER), optical filters and mirrors which select and separate emission wavelength towards the appropriate detectors FACSJazz Becton Dickinson Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Fluidic system Sample under pressure Cells suspension Sample pressure hydrofocalization Laminar flow Psa low sheath fluid under pressure Sample under pressure Cells suspension Carry away Psa high sheath fluid under pressure Sheath fluid flow induces a fast and regular acceleration to cells and forces them to be aligned in order to be analyzed one by one Flow Cell Nozzel Flow cell Light excitation Light excitation Psa Cell flow Separation Carry vein Weak focalization of cells Tiny distribution Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Ps Important pressure difference Fast speed of passage Weak pressure difference Slow speed of passage Important focalization of cells Alignment Light excitation (laser(s), lamp) Psa Ps Large distribution Psa = pression sample Ps = pression sheath Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Optical system Optical system The cytometer records the behaviour of the cell trough the laser beam, by measuring : Fluorescence emission (« Fluorescence Light » (FL)) Spontaneous (autofluorescence) Associated to free or bound fluorochromes Light scattering (informs on the morphology and cell structure) Detection at 90° granularity: SSC or SS or 90LS Fluorescence are emitted in all directions and are always in another color than the excitation laser Detection at small angles size : FSC or FS or FALS Light excitation Invitrogen Invitrogen - Light scattering at small angles (Forward Scattered Light, FSC) represents the light incidence angles from the LASER and is directly proportional to the particle size and area (relative measure of size) - Light scattering at large angles (Side Scattered Light, SSC) measures the reflected light and depends on granularity and complexity of the particle (relative measure of Color depends of the fluorescent probe nature Invitrogen Induced fluorescence from different fluorochromes is separated by an optical filters plot. The selection filters depends on the Excitation and emission wavelengths granularity) Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Optical system Optical filter Filters and dichroic mirrors are part of optic elements. They realize the separation of channels and select wavelength FSC signals are received by a photodiode. SSC signals and fluorescents emissions are collected and diverted to photomultipliers tubes (PMT). Each PMT have an optic filter which allow to detect specific wavelength Each fluorochrome is detected in a unique fluorescence optic channel: emitted light follows a specific path from the “interrogation point” to the detector Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Optical filters are materials which absorb specific wavelength and transmit others Incidence ray Transmitted light Absorbe light When filter and incidence light make a 45° angle the not absorbed wavelengths are reflected Dichroic mirrors Reflected light Transmitted light Incidence ray 45° Reflection Transmission Absorption Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Optical filter Optical filter Different filters types used in CMF Different filters types used in CMF 525 500 475 l <475 l <500 Transitions between absorption and transmission are not perfect LP filter 100 100 50 0 wavelength l 50 0 100 50 0 50 0 SP 520 LP 520 BP 500 BP 500/40 500 +- 20 100 50 0 Band Pass filter 100 example BP filter % de transmission % de transmission % of transmission SP filter 100 % de transmission 640 570 l >500 Long pass filter Short Pass filter % de transmission l >525 500/50 LP500 l <500 480 460 Band Pass filter % of transmission Long pass filter SP500 l >500 Short Pass filter wavelength l 480 50 440 0 wavelength l 480 520 560 wavelength l 600 640 440 480 520 560 600 wavelength l 640 440 Service Imagerie Cellulaire et Cytométrie en Flux 20 480 20 520 520 560 600 wavelength l 640 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Optical filter Optical system Band Pass 525/40 Different filter types used in CMF 505 BP 525/40 525 +- 20 nm 545 Ex : Optical bench Ligth sources Light source 20 20 460 500 Dichroïc Long Pass 550 540 580 wavelength l 620 680 BD LSR cell analyzers Invitrogen Light source 460 500 540 580 wavelength l 620 680 BC Gallios BC MoFlo XDP Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2012 Optical system Electronic system In order to measure the optical signals they have to be converted in electric signals (volts). The choice of the detector depends on the photon number Impulse formation Flow 10 Laser Diodes 1v 1e- Volt Laser Volt Detect intense signals (ex : size parameter) Photomultipliers Tubes (PMT) 1v ne- Area (A) Height (H) Photons detectors The electrical signals created by light emission are called impulsions. Their amplitude values lies between 0 and 10 volts 0 Width (W) High sensitivity (ex : structural and fluorescent parameters detector) used for weak signals Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Time (µ Seconds) Laser Time Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Electronic system Electronic system electrical impulsion (volt) has to be transformed into numerical data by a digital analogical converter (ADC) 1024 0 0 10 1024 0 0 10 1024 0 0 10 1024 0 0 10 1024 0 0 2 450 1024 ex : An ADC of 10 bits (210) enables 1024 values Conversion Light excitation 3 4 Linear scale Time 0 0 Photomultiplier or Logarithmic 5 Amplification Flow 450 900 450 450 Service Imagerie Cellulaire et Cytométrie en Flux Relative light intensity Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Data acquisition Data representation The cytometer stores all parameters of every cell : List mode (LMD) SSC FL1 FL2 FL3 Time (µsec) 1rst 119 2de 124 65 541 797 669 1 86 120 842 669 3rd 1 223 252 574 837 730 2 4th 144 71 69 807 686 1 .... …. …. …. …. …. …. Last 112 87 574 83 655 1 UR LL LR + Other two parameters representations Cells number UL - Relative light intensity Three-dimensional plot Advantage : everything could be recalculated 842 Two parameters histogram Histogram Cells number FSC 900 Arbitrary units etc Each mesurement from each detector is called to as a parameter Cells analysis 900 Cells number 10 10 1 Convert into binary signal by the ADC, signals are processed individually arbitrary units Intensity (volts) Cells 83 120 574 Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Threshold and trigger parameters Data analysis Trigger parameters Regions permit to Electronic treatment of the signal Isolate groups of interest, Threshold Often done on the size, it allows to reduce the number of events without interest to not saturate the system Measurement of amplification 0 10 Time (microsec.) Volt 10 Monocytes SSC Threshold level FSC Lymphocytes 0 10 Time (microsec.) SSC Measurement of amplification Threshold level 0 statistic calculations Granulocytes Volt 10 0 better discrimination by coloration, FSC Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Data analysis Data analysis Gates A few examples Permit to combine logical regions, to define subgroups and to restrict the analyze on interesting cells signals to characterize them (statistics) Percentage R1 : 68,94% G2/M R2 : 30,96% R2 240 overlay R3 R3 : 0,05 10 3 events Counts R2 mean : 333,75 R2 R2 : 1,29 10 4 FL2-Log_Height Comp 360 R1 R1 R1 R2 R1 mean : 6,45 Ungated G0/G1 10 5 481 10 2 120 S 10 1 Gated Doublets 0 100 2C R5 R5 : 0,72 R4 R4 : 97,93 R1 and R2 101 102 103 FL1-Log_Height 104 105 10 0 100 101 102 103 FL1-Log_Height Comp 104 105 4C Booleans equations …etc. AND OR NAND NOR Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Doublet elimination A. Munier-01/2013 Signal diffraction, refraction and reflection Refraction (dead or alive) Laminar flow Diffraction (size) Laser G2 or M Doublet G1 2 x G1 events G1 4n Temps 1 Area 1 2 2 Height Peak 1 2 1 (relative value) 1 Refraction is principally between 2 and 12° angles: FSC Reflection (cell surface, nucleus, granules…) 2n Integral Area (relative value) G2 2 Peak Intensity Fluorescence Excitation light G1 Reflected light Propidium Iodide 2 Refracted light Excitation light Diffraction is principally between 0 and - 4° angles: FSC Excitation light Reflected light Reflection is principally at 90° : SSC Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Physical properties A. Munier-01/2013 Discrimination between living and dead cells Deaths Cells Granulocytes Homogeneous cell size Living cells Forward scatter Heterogeneous cell size Monocytes Forward scatter Lymphocytes Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Fluorochrome characteristics Brightness fluorochromes Fluorochrome Molecule induces an electronic transition which then induces fluorescence. Associated to a macromolecule, it is called fluorophore Extinct coefficient multiplied by quantum output Excitation spectrum are compatible with the light source ABS EM CD8-PerCP wavelegnth (l) Quantum output CD8-FITC CD8-ECD CD8-APC Ratio between the number of emitted photons and the number of absorbed photons Extinct coefficient e Measurement of a reference fluorophore at a wavelength at 1M in a 1cm tank. (the more the value the more the fluorescence is elevated) Signal stability CD8-PC5 CD8-PC7 Signal stochiometry Service Imagerie Cellulaire et Cytométrie en Flux CD8-PE Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Choice of fluorochromes A. Munier-01/2013 Spectral overlapping …BUT for multicolor is very difficult… The excitation spectrum are compatible with your system Use brightest fluorochromes for least expressed proteins Use dimmest fluorochromes for most highly expressed proteins Choose association of fluorochromes to avoid overlaps... …hence limit spectral overlaps between fluorochromes and… calculate compensation… two methods for compensation : Medians method (manual) Slope method (automatic with new generation system) Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Spectral overlapping A. Munier-01/2013 Medians method (Manual compensation) Intensity A contamination percentage of a fluorochrome by another B FL1 FL2 530/40 580/30 Tubes must contain negatives cells and monomarked cells (or beads) Photomultipliers voltage has to be high enough, allowing to have negatives cells in first decade Gate must include positives and negatives autofluorescence (ex gate on lymphocytes). A cells of a same Adjust the compensation so as to positives and negatives cells, have same median The calculated shift must be then entered in the system This procedure must be done on each fluorophore (do not forget to reset all parameters Photomultiplier FL2 detects FITC fluorescence (A) Photomultiplier FL1 detects PE fluorescence (B) Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Compensation – Medians method Compensation A label (not a color) is attributed to each detector. The compensation method consists in removing a percentage from a signal to another signal 10 5 Fluo1 Fluo2 Counts 10 2 Fluo2 -PE 736 402 552 301 Counts PE - FL2-Log_Height 10 3 368 Compensation etc. PMT ADC 201 184 100 10 1 R4 10 0 100 10 5 Fluo1 Fluo2 101 102 103 104 FITC - FL1-Log_Height 101 102 103 104 FITC - FL1-Log_Height 0 100 105 101 102 103 104 PE - FL2-Log_Height 105 105 digital signal R3 R2 Fluo1 -FITC 10 4 FL2-Log_Height Comp PE - FL2-Log_Height 580/30 10 3 Counts 530/40 0 100 R5 10 2 Fluo2 -PE 736 402 165 552 301 123 Counts Counts Wavelength Intensity Fluo1 -FITC 10 4 580/30 Intensity 530/40 Analog signal R3 R2 368 PMT 201 82 Compensation etc. ADC 100 41 184 10 1 R4 10 0 Wavelength 100 0 100 R5 101 102 103 104 FITC - FL1-Log_Height FL1-Log_Height Comp 105 101 102 103 104 FITC - FL1-Log_Height 105 0 100 101 102 103 104 PE - FL2-Log_Height FL2-Log_Height Comp 105 Compensation PE-%FITC Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux Compensation Log 4 decades A. Munier-01/2013 From C. Aït Mansour - Miltenyi Compensation Log 5 decades hyperLog Make compensation settings with bright fluorophores Do not touch values of PMTs after compensation settings No compensation Compensation Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 From C. Aït Mansour - Miltenyi A. Munier-01/2013 Required controls Dilution of Antibodies Required controls Titrate antibodies Example 5 colors N° Tube FITC PE PE-TR PE-CY5 PE-CY7 1 expérimental CD3 CCR7 CD45 RO CD27 CD8 2 non coloré - - - - - 3 Ctrl isotypique t t t t t 4 monomarquage CD3 -/t -/t -/t -/t 5 monomarquage -/t CCR7 -/t -/t -/t 6 monomarquage -/t -/t CD45 RO -/t -/t 7 monomarquage -/t -/t -/t CD27 -/t 8 monomarquage -/t -/t -/t -/t CD8 Secondary Ab 1/100 Secondary Ab 1/50 Control with the same isotype and the same dilution as the specific antibody Primary Ab 1/25 1/50 1/100 Useful controls : FMO (Fluorescence minus one) 9 Ctrl régions - CCR7 CD45 RO CD27 CD8 10 Ctrl régions CD3 - CD45 RO CD27 CD8 11 Ctrl régions CD3 CCR7 - CD27 CD8 12 Ctrl régions CD3 CCR7 CD45 RO - CD8 13 Ctrl régions CD3 CCR7 CD45 RO CD27 - 1/200 Cytométry 69A : 1037-1042 (2006) Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Spreading data Unstimulated control Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux Cytométry 69A : 1037-1042 (2006) A. Munier-01/2013 Antigens/ receptors Excitation Emission Nucleic acids Excitation Emission AMCA 354 442 DAPI, Hoechst 33258 354 442 A-T bases Pacific blue 410 455 Hoechst 33342 354 463 A-T bases, vital FITC 495 520 Phycoérythrine 495, 532, 560 575 PE-Rouge Texas* 495 620 PE-Cyanine 5° 495 670 Allophycocyanine 650 PE-Cyanine 5.5 488, 532, 560 PE-Cyanine 7 495 765 APC-Cy7 650 785 Sytox blue 430 480 Intercalate, viability Cell trace calcein violet AM 405 450 hydrophobic Red 613, ECD YO-YO 470 510 ADN Tricolor, quantum red. Visible light sensibility Acridine Orange 500 530, 640 ADN/ ARN 660 TO-TO 510 533 ADN 720 Chromomycine 430 570 G-C bases Sensible to pH Pyronine Y 540 570 ARN Propidium Iodure 536 (360 et 488) 617 Intercalate : ADN and/or ARN, viability EMA, ethydium monoazide 510 600 Viability, covalent/ photo-achievable 7 A-actinomycine D 478 660 G-C bases, viability CY7 could be altered by the fixation Insensible to pH, soluble in water Alexa : 350, 405, 430, 488, 532, 568, 594, 610, 633, 647, 660, 680, 700, 750 °PE/CY5 fixed monocytes and B lymphocytes of mice SJL, AKR, NOD Rappel PE-CY5 excited at 488 nm by the emit energy transferred from PE but also by the laser 633 nm via CY5, compensations could be elevated or difficult to realize so try to replace PE-CY5 by PECY5.5 or PE-CY7 in a combination with APC Amine reactive dyes : an effective tool to discriminate live and dead cells in polychromatic flow cytometry. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roderer M,. J Immunol Metghods. 2006 Jun 30;313(1-2):199-208 Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Cellular functions Excitation Monochlorobimane 360 420 glutathion INDO-1-AM 335 485/ 410 Free/ bound Ca2+ FLUO-3 506 530 Free non fluo/ bound Ca2+ fluo A. Munier-01/2013 Emission Fura Red 480 660 Free fluo/ bond CA2+ non fluo DIOC4 à 6, JC-1, CMXRos Rh 123 484 510 510 530 Mitochondrial potential NAO (nonyl acridine orange) 489 525 BCECF 500 520/ 620 Mitochondrial mass membrane SNARF 518 575/ 670 pH6/ 7,5 CFSE 495 530 pH6/ 9 FDG 490 520 Cellular proliferation 490 513 B-galactosidase Apoptosis Excitation Emission Phiphilux 488 530 Caspases-3 Annexin-V FITC 495 520 Phosphatidyl-serine dUTP-FITC 495 520 3’-OH DNA ends YO-PRO 495 520 ADN SYTO-17 490 675 ADN ECFP 433 475 EGFP 480 505 EYFP 513 527 490, 550 590 mRFP 580 610 Spectrum excitation U.V : Hoechst, DAPI 488 : IP, 7-AAD Fluoresceine Diacetate FDA DsRed Ex : Nucleic acid dies 633 : TO-PRO3 Permeant Hoechst Vybrant DyeCycle Violet stain Specificity Non : IP A-T : Hoechst C-G : 7-AAD, Chromomicyne Toxicity Hoechst DRAQ5 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Cell Flow Sorting Continuous jet sorting : principle Partec System Becton System Cell Flow sorting Fluid Separate cells in a flux according to their properties Piezo-electric valve Fluorescence-Activated Cell sorting FACS is a trademark of Becton Dickinson Cells path when opened valve (unsorted cells) Collection tube Cells path when closed valve (sort) Center collection tube : sort Off center collection tube : unsorted Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Cf C. Jayat Atelier INSERM 103 Sort “jet-in-air” Electrostatic sorting nozzle Droplet formation 1- Adjust sample and liquid sheath pressure. 2- Induce an amplitude variation and a variable frequency on the flow stroboscope Drop delay Time needed for one cell to go from the analyze point to the droplet formation Target cell identification Deviation plates To analyze cells and to define sorting windows Deflected jets Droplet charge The drop charge determines the applicable tension on a droplet. Charged droplet will be deviated from the normal path Droplet deviation Waste bin An electrostatic field deviate the droplet Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Cell sorting “jet-in-air” : principle Sort strategyB A Electric impulsion Variable position of the cell in the flow (coincidence notion) nozzle Time 0 Laser beam Detector Time 1 Drop Delay Intersection flux / laser Time 0 Time 1 ? Last attached droplet (DGA) A sorting envelop is defined depending on the direction needed for cell sorting Deflection charged plate + + + Purity 1 droplet Detection of coincidence ON Purity/ Output 2 droplet Detection of coincidence ON Output/ Purity 3 droplet Detection of coincidence ON Output 3 droplet Detection of coincidence OFF - Waste bin 2 Collecting tubes support Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 3 1 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Application field Studies Morphological analysis size, numeration, cellular viability, Antigenic analysis (membrane and cytoplasme) phenotype of blood cells, hematology, leukemia's, antigenic quantification, allergologic tests, prenatal diagnosis, specificity and antibody titration... Fundamental and applicative research Medicine (hematology, immunology, cancerology …etc), Industry: food processing, environment, cosmetology, pharmacology Functional analysis cellular signalization, metabolic activity, membrane integrity, proteins and genetic expression, ionic flux, intracellular PH... Nucleic acid analysis DNA and RNA content, cellular cycle, proliferation, apoptosis, cellular viability... and toxicology. Cell sorting physiological criterions (fluorochromes), fluorescents telltale genes (GFP…), phases of the cellular cycle, males or female spermatozoids... Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Flow cytometry and safety DNA/ cell cycle Biological and chemical risks G1 Phase G0/G1 21,8% G2+M 33,6% Linear markers give an indication but it is just an estimation Cell Number Electrical risks 44,6% S LASER risks Phase S Phase G2 + M G1 41,6% S 28,8% G2+M 29,6% Aerosols risks Propidium Iodure Mathematic models without being perfect Sound risks are more precise MultiCycle Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Essential conditions for DNA content analysis Cell cycle Identification of mitosis cells Apparition of the CyclineA2 during the cycle IgG1 Optimal use of the cytometer (marble of calibration, DNA beginning of the fluorescence, correct voltage) Cyclin A2 FITC Passing time : 200 events per second Coefficient of variation peak G0G1 < 5% Weak fragment proportion (<20%) Ctrl Eliminate aggregates Propidium Iodide 10 000 cells minimum to calculate a cycle S G2/M Have diploid cells as reference (ex : chicken red blood cells…) Propidium Iodide MPM2 Alexa 488 fluorescence Utilization of a valid staining method for DNA Cell Number G0/G1 M G2 DNA content PI fluorescence J. Sobczak UMR 7622 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux I. Gasnereau UMR 7622 A. Munier-01/2013 Modulation of the cell cycle Cell cycle Divers inhibitors effects of topo-isomerases (treatment during 16 hours) DNA peak a tumorous cell line treated by antimitotic agent % of cells in G2: R1 R1 Cell number R1 Reference: 16% R1 2c drug 0.1: 26% drug 0.3: 50% drug 0.9: 83% 1c Asynchrones cells Camptothecine 10 mM Etoposide 250 nM 4c ICRF-193 10 mM Service Imagerie Cellulaire et Cytométrie en Flux J. Sobczak UMR 7622 A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux Agnès Chassevent - 2009 Paul Papin Center - Angers Cell proliferation A. Munier-01/2013 Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) Click-it EdU follows a protocol of Aldehyde fixation and detergent permeabilization Phase S G2 + M G1 + G0 0 • Fix for 15 minutes, wash • Permeabilize for 30 minutes, wash • Incubate in click labeling cocktail for 30 minutes, wash • Optimal : incubate with cell cycle stain for 15-30 minutes • Analyze 200 400 600 Control cells : EdU cells 800 EdU-incorporated 1000 FL3-H Service Imagerie Cellulaire et Cytométrie en Flux J. Sobczak UMR 7622 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Analysis of cell division Clinical study of MDR* *MDR Resistance MultiDrogue Proliferation of T lymphocytes after 4 days of stimulation D4 N°1 N°2 N°3 D6 events D0 with verapamil (carboxyfluoresceine diacetate, succimidyl ester) CD56 CFSE without verapamil Rhodamine 123 A. Saoudi Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Drénou, B Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Effect of transfected gene on cell cycle Ki-67 /DNA 200 transfected gene 150 32% of cells Ki-67 + 38% # Cells 4 10 100 6.35 0 200 400 600 FL4-H 800 Ki-67 FITC GFP FL1-H 0 2 1000 Hoechst 33342 10 5000 1 10 93.2 Ki-67 FITC 50 3 10 # Cells 4000 3000 0 10 0 200 400 600 FL4-H Hoechst 33342 800 1000 2000 20% 1000 Propidium Iodide (DNA) 0 0 200 400 600 800 1000 Service Imagerie Cellulaire et Cytométrie en Flux Derek Davies, Cancer Research UK A. Munier-01/2013 Propidium Iodide (DNA) Service Imagerie Cellulaire et Cytométrie en Flux Agnès Chassevent - 2009 Paul Papin Center – Angers A. Munier-01/2013 Apoptotic event Apoptotic event Exposure of phosphatidylserine to external face cytoplasmic membrane intracellulare Ca++ increase Indo1 emits blue fluorescence when it is free Indo 1 emits purple fluorescence when it is binding Ca++ They measure ratio indo bind/indo free according to time Propidum iodide T human lymphocytes non stimulated T human lymphocytes antigen timulated Annexin V- FITC Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux S. Valitutti A. Munier-01/2013 Apoptotic event DNA Fragmentation TUNEL pic sub-G1 (TdT-mediated X-dUTP nick end labelling) 2n 4n Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 J.F. Mayol – CRSSA - Grenoble Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Stem cells DNA/ Cell cycle - Sorting 600 Side Population (bone marrow of mouse) Hoechst 33342 (laser UV or violet) emission : blue AND red Apo G0/G1 S G2+M G0/G1 events Muscle Hematopoietic Apo. S G2+M 0 Adipocytes 0 Osteoblastes 33% 47% 14% 6% Propidium Iodide Overlay after sorting 1024 G0/G1 events Endotheliales S G2+M Detection based on efflux Hoechst 33342 Propidium Iodide Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux M.C. Gendron A. Munier-01/2013 Multicolor analysis Multicolor 6 coulors 26 population CD3 FITC CD38 PE CD45 PerCP-C5.5 CD4 PE-Cy7 CD19 APC CD8 APC-Cy7 after sorting Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Multiplex A. Munier-01/2013 Detection of small events Ex : Cytometry Bead Area – BD Biosciences assays on beads New cytometers Different Intensity of fluorescence read on FL3 aIL-8-PE FL3 - Beads FL3 - Beads aIL-1b-PE aIL-6-PE FL2 - PE Detection of small particles (0.2 µm) according to their size and their fluorescences and of intensity of their fluorescence aIL-10-PE aTNF-a-PE aIL-12-PE Ann Biol Clin, vol. 67, n°4, juillet-aout 2009 sensitivity, optic, electronic, informatic Fluorescences probes FL2 - PE Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Detection of microorganisms Detection of microorganisms Autofluorescence Resolution of multiple small species by forward and side scatter Chlorophylle Phycoerythrine Phycocyanine Picoeucaryotes Synechococcus Prochlorococcus Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Marine Biology Medical Biology Crucial role of microorganisms in the functioning of earth’s biosphere No Antibiotic Antibiotic added FS vs SS FS vs SS Examples of phytoplancton population analysis coming from different coastal ecosystems with each cell being identified by their light scattering properties (size) and by their red chlorophyllfluorescence excited with a 488 nm laser. Light scatter cytograms of E.coli cells before and after addition of ampicillin. Data obtained from, Bio-Rad, USA. Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Food Biology Plant Biology DNA in plants Detection of Listeria in milk products with CMF using fluorescent antibodies automatical analysis methods in milk products Listeria monocytogenes Not just pathogenics ... Viability studies of beer yeast used in australian brewing industry Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 S.J. Ochatt Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Supplementary methods A hybrid Flow cytometry Microscopy Quantification +++ +/- Discrimination individual individual Visualization - +++ Photobleaching - +++ Reproductibility +++ + Observed events ∞ Some hundreds Cell sorting +++ +/- Complementarity YES YES The ImageStream is a high-speed automated microscope that captures images in flow (low rate) detector Spectral decomposition element cells in flow Brightfield illuminator autofocus laser ImageStreamX Amnis Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Technological innovation Velocity detector A. Munier-01/2013 Technological innovation LEAP is an imaging cell sorter (shooting laser) Attune a cytometer an acoustical focusing Attune Life Technologies Schematic of acoustic focusing principle. LEAP Cyntellect Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Technological innovation Perspective CyTOF : metal-labeled cells are introduced individually into an Inductively Coupled Plasma Cells are atomized and ionized in an argon plasma Atomic ions are extracted into the ion optics and Time-Of-Flight region They are separated by mass and counted Quantum Dots, are semiconductors whose electronic characteristics are closely related to the size of individual crystal. Excitation par UV Analysis of up to 100 stable isotope labels in a single cell No overlap between detection channels, no need to compensate No autofluorescence An unlabeled cell is invisible to the system 1000 cells/ sec Efficiency of the cell intruction system 20-30% Data output in text and FCS standards CyTOF® system DVS Sciences Bandura, D.R., et al. Anal Chem 81 (16): 6813-6822, 2009. The smaller dot, the closer it is to the blue of the spectrum, and the larger dot, the closer to the red. Dots can exciting by U.V or another laser. Quantum Dot Corporation Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Recent technological developments Conclusion Sensitivity increase and multiparameters « ELISA on beads » for detection and quantification of several products in the same sample CMF is a technology turned to the polychromy Panel of cytokines in plasma Receptor in supernatant or in cell lysate RNA messengers « microarrays » on beads Sensitivity increase and number of analyzed parameters open prospects into studies of microparticules Current technological developments QuantumDots (nanocristaux) Service Imagerie Cellulaire et Cytométrie en Flux Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 Documentation Documentation Books and journals Articles La cytométrie en flux. X. Ronot, D. Grunwald, J.F. Mayol & J. Boutennat. Tec et Doc. Lavoisier. Paris. 2006. Hoffman RA,Wang L, Bigos M, NolanJP : NIST/ISAC standardization study : variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads. Cytometry . 81A : 785-796. (2012) Practical flow cytometry. Shapiro H.M. Zbigniew Darzynkiewicz : Critical Aspects in Analysis of Cellular DNA Content. Curr Protoc Cytom. 2011 April; CHAPTER: Unit–7.2. (2011) 4ème édition. 2003. Wiley-Liss ed. Roderer : Optimizing a multicolor immunophenotyping assay. 485. (2007) Cycle cellulaire et cytométrie en flux. Grunwald, J.F. Mayol & X. Ronot. Tec et Doc. Lavoisier. Paris. 2010. Cytometry : http://eu.wiley.com/WileyCDA/WileyTitle/productCd-CYTO.html Service Imagerie Cellulaire et Cytométrie en Flux Clin Lab Med 27 : 469- Roederer : Quantum dot semiconductor nanocrystal for immunophenotyping by polychromatic flow cytometry. Nat Med. Aug ; 12(8) : 972-7. (2006) Maecker HT, Trotter J : Flow Cytometry Controls, Instrument Setup, and the Determination of Positivity.. Cytometry 69A : 1037-42. (2006) Perfetto, S. P., Chattopadhyay, P. K. and Roederer, M. : "17-Color Flow Cytometry: Unraveling the Immune System." Nat Rev Immunol 4: 648-655. (2004) Robert P. Wersto, et al : Doublet Discrimination in DNA Cell-Cycle Analysis. Cytometry. 46A : 296-306. (2001) Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 A. Munier-01/2013 SPACE Documentation Web cytometry Association Française de Cytométrie : http://afcytometrie.fr OCEAN International Society for Analytical Cytology : http://isac-net.org/ Life technologies : http://www.invitrogen.com/site/us/en/home.html Analyse cellulaire Flow cytometry resources flow cytometry tutorials EARTH List of discussion TARA Liste de discussion : http://www.cyto.purdue.edu/ Free software WEASEL : http://www.wehi.edu.au/cytometry/WEASEL.html-net.org/ Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013 Service Imagerie Cellulaire et Cytométrie en Flux A. Munier-01/2013
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