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). Visit bdbiosciences.com for more information. 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. Class 1 Laser Product. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2014 BD 23-16934-00 BD Biosciences bdbiosciences.com
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