Immunophenotyping Cancer Cells Using Flow Cytometry
Cancer Biology Applications on the BD Accuri™ C6
Features
Immunophenotype cancer cells based on surface markers
Screen cancer cells for expression of surface proteins
Characteristic
MDA-MB-231
MDA-MB-468
MCF-7
Tumor
classification
Epithelial breast
adenocarcinoma
Epithelial breast
adenocarcinoma
Epithelial breast
adenocarcinoma
Derivation
Metastatic site
(pleural effusion)
Metastatic site
(pleural effusion)
Metastatic site
(pleural effusion)
Enrichment
Cancer stem
cell phenotype
(CD44+CD24–)
None
Small
subpopulation
(CD44+CD24–)
Q1-UR
1.0%
Q1-LL
0.5%
Q1-LR
0.0%
10 4
10 5
10 6
10 7.2
Q1-UL
0.1%
Q1-UR
99.2%
P1
65.1%
5,000,000
FSC-A
10,000,000 14,096,928
10 5
10 6
10 7.1
D04 468, CD44, CD24
Gate: (P1 in all)
10 4
FL4 CD44 APC-A
2,000,000
10 3
FL2 CD24 PE-A
D04 468, CD44, CD24
Gate: [No Gating]
199,135
10 5
10 1.4
10,000,000 14,096,928
0
SSC-A
Q1-UL
98.5%
10 4
10 2.5 10 3
FL4 CD44 APC-A
FSC-A
4,000,000
5,883,678
5,000,000
10 2.5 10 3
2,000,000
P1
61.3%
199,135
MDA-MB-468
B06 231, CD44, CD24
Gate: (P1 in all)
10 6
4,000,000
10 7.1
B06 231, CD44, CD24
Gate: [No Gating]
0
SSC-A
MDA-MB-231
5,883,678
Table 1. Breast cancer cell lines used in immunophenotyping examples1,2
Q1-LL
0.4%
10 1.4
Q1-LR
0.2%
10 3
10 4
10 5
10 6
10 7.2
FL2 CD24 PE-A
Figure 1. Immunophenotyping breast cancer cell lines for cancer stem cell
markers
MDA-MB-231 and MDA-MB-468 cells (human epithelial breast adenocarcinoma;
ATCC) were disassociated with BD™ Accutase™ Cell Detachment Solution (Cat.
No. 561527) and stained with BD Pharmingen™ Mouse Anti-Human CD24 PE and
BD Pharmingen™ Mouse Anti-Human CD44 APC (Cat. Nos. 555428 and 559942).
Data was acquired on a BD Accuri C6 and analyzed using BD Accuri™
C6 software. Results: Cells were initially gated based on light scatter properties
(left plots). As expected, MDA-MB-231 cells (upper plots) expressed a cancer stem
cell phenotype (CD44+CD24–) while MDA-MB-468 cells (lower plots) expressed both
CD24 and CD44. Gates were drawn based on isotype controls
(data not shown).
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
A personal flow cytometer in the lab provides many advantages
for cell and cancer biology studies. When cells are ready for
analysis or rare tumor samples arrive, it’s crucial to have a flow
cytometer at hand, ready to go. This data sheet shows the kind
of rich data you can generate using the BD Accuri™ C6 personal
flow cytometer for two kinds of cancer biology studies.
Experiment 1: Immunophenotyping of cancer cell lines
Experiment 1 demonstrates immunophenotyping of MDAMB-231 and MDA-MB-468, two of the breast cancer cell lines
shown in Table 1. Immunophenotyping is one of the foremost
applications of flow cytometry because of its ability to recognize
different cell types based on the expression of surface and
intracellular proteins.
Figure 1 shows the results when MDA-MB-231 and MDAMB-468 cells were tested for expression of CD24 and CD44,
two known cancer cell markers. With two lasers and four
fluorescence detectors, the BD Accuri C6 could have tested for
expression of two or more additional surface or intracellular
markers as well.
Experiment 2: Surface marker screening of a cancer cell line
If you don’t yet know which proteins are typically expressed by
a subpopulation of interest, BD Lyoplate™ cell surface marker
screening panels provide a comprehensive and efficient solution
for profiling cancer cells for hundreds of human or mouse cell
surface markers by flow cytometry. Deciphering the cell surface
proteome enables researchers to define strategies for the analysis
and isolation of targeted cells from heterogeneous populations
for functional studies, drug screening, and in vivo animal
studies.3,4
Both the human (Cat. No. 560747) and mouse (Cat. No. 562208)
screening panels contain three plates. Each well contains
lyophilized, purified antibody to one cell surface marker or
isotype control. The process is illustrated in Figure 2.
Figure 3 shows the results when MCF-7 breast cancer cells were
analyzed for surface marker expression using the BD Lyoplate™
Human Cell Surface Marker Screening Panel (Cat. No. 560747).
The heatmap summarizes the expression of selected markers,
from those expressed almost universally to those expressed rarely
or never. The plots show different patterns of expression for
selected markers.
Easy to use, simple to maintain, and affordable, the BD Accuri
C6 personal flow cytometer is equipped with a blue laser, a red
laser, two light scatter detectors, and four fluorescence detectors.
A compact design, fixed alignment, and pre-optimized detector
settings result in a system that is simple to use. A nonpressurized
fluidics system enables kinetic measurements in real time. For
walkaway convenience, the optional BD CSampler™ accessory
(Cat. No. 653124) offers automated sampling from 24-tube racks
or multiwell plates.
Immunophenotyping Cancer Cells Using Flow Cytometry
100%
Prepare a single-cell
suspension and
aliquot into three
96-well plates
Wash
Secondary antibody
Wash
Fix
Wash
Transfer
recontituted
antibodies to
plates with cells
Reconstitute
BD Lyoplates
Collect data
CD49c
99.95
CD166
99.31
CD47
99.31
CD44
99.16
CD24
99.09
CD49f
84.17
CD104
79.71
CD107a
74.49
CD15
67.87
CD220
66.07
CD10
56.21
EGFR
49.89
CD29
36.00
CD146
34.10
CD100
24.36
CD161
19.90
CD91
15.36
CD268
11.92
CD33
7.43
CD120a
5.54
CD66f
0.00
0%
Analyze data
34.10
CD100
24.36
CD161
19.90
CD91
15.36
CD268
11.92
CD33
7.43
CD120a
5.54
CD66f
0.00
0
10 1
10,685,489
FSC-A
10 2
10 3
10 4
10 5
10 6
10 7.2
10 1
E
200
Count
50
Count
10 1
10 2
10 3
10 4
10 5
10 6
10 7.2
10 4
10 5
10 6
10 7.2
G10
Gate: P1
M1
19.9%
M1
0.3%
0
0
0%
10 3
FL4 CD44 Alexa 647-A
F
50
M1
34.1%
10 2
FL4 CD24 Alexa 647-A
E11
Gate: P1
150
100
500
CD146
D
400
36.00
5,000,000
E02
Gate: P1
200
49.89
CD29
Count
EGFR
599,187
0
56.21
600
66.07
CD10
M1
98.7%
400
67.87
CD220
Count
CD15
M1
98.9%
200
74.49
E04
Gate: P1
0
79.71
CD107a
200
84.17
CD104
150
99.09
CD49f
Count
CD24
P1
95.6%
50
99.16
C
C06
Gate: P1
100
99.31
CD44
SSC-A
99.31
CD47
B
C06
Gate: [No Gating]
1,000,000 2,000,000
CD166
A
0
99.95
100
100%
CD49c
3,595,118
Figure 2. BD Lyoplate surface marker screening workflow
10 1
10 2
FL4 CD146 Alexa 647-A
10 3
10 4
10 5
10 6
10 7.2
FL4 CD161 Alexa 647-A
10 1
10 2
10 3
10 4
10 5
10 6
10 7.2
FL4 CD66f Alexa 647-A
Figure 3. Surface marker screening of breast cancer cells
A single-cell suspension of MCF-7 cells (human breast adenocarcinoma; ATCC) was prepared using BD Accutase Cell Detachment Solution (Cat. No. 561527). Fifty
million cells were aliquoted in three 96-well plates (~180K cells/well) and stained with the BD Lyoplate Human Cell Surface Marker Screening Panel (Cat. No. 560747).
After staining, cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). Plates were sealed and stored at 4°C. Cells were acquired within 3 days using the
BD CSampler accessory (Fast speed, 1 wash cycle, 2 agitation cycles every 4 wells), which processed each plate in 2.5–3 hours. Cells were analyzed using BD Accuri C6
software. Results: (A) Cells were initially gated based on light scatter properties. (B-F) M1 gates were drawn based on isotype controls. (B, C) Almost all cells expressed
both CD24 and CD44. (D) Cells expressed varying levels of CD146. (E) CD161 showed a bimodal distribution. (F) Almost no cells expressed CD66f. A heatmap (left)
summarizes the expression of selected markers tested.
Ordering Information
References
Description
Cat.No.
BD Accuri™ C6 Flow Cytometer System
653118
BD CSampler™ Automated Sampling System
653124
BD Pharmingen™ Mouse Anti-Human CD24 PE
555428
BD Pharmingen™ Mouse Anti-Human CD44 APC
559942
BD Lyoplate™ Human Cell Surface Marker Screening Panel
560747
BD Lyoplate™ Mouse Cell Surface Marker Screening Panel
562208
BD™ Accutase™ Cell Detachment Solution
561527
BD Cytofix™ Fixation Buffer
554655
1. M
urohashi M, Hinohara K, Kuroda M, et al. Gene set enrichment analysis
provides insight into novel signaling pathways in breast cancer stem
cells. Br J Cancer. 2010;102:206-212.
2. S heridan C, Kishimoto H, Fuchs RK, et al. CD44+CD24– breast cancer
cells exhibit enhanced invasive properties: An early step necessary for
metastasis. Breast Cancer Res. 2006;8:R59.
3. S ukhdeo K, Paramban RI, Vidal JG, et al. Multiplex flow cytometry
barcoding and antibody arrays identify surface antigen profiles of
primary and metastatic colon cancer cell lines. PLoS One. 2013;8:e53015.
doi: 10.1371/journal.pone.0053015
4. L athia JD, Li M, Sinyuk M, et al. High-throughput flow cytometry
screening reveals a role for junctional adhesion molecule a as a cancer
stem cell maintenance factor. Cell Rep. 2014;6:117-29.
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