Enhanced Cell Cycle Studies Using Multicolor Flow Cytometry: Cell

Enhanced cell cycle studies using multicolor flow cytometry Cell biology applications on the BD FACSCelesta™ flow cytometer

Features With up to three lasers and 12 fluorescence detectors, the BD FACSCelesta™ flow cytometer provides a flexible tool for cell Obtain enhanced cell cycle data at the single-cell level biologists to complement traditional Western blot analysis with rich Reduce spectral overlap and minimize compensation by cell cycle data and immunophenotyping. While Western blot analysis spreading fluorochromes across three lasers analyzes populations in bulk, multiparameter flow cytometry enables the quantitative analysis of populations at the single-cell level. It Combine multiple cell biology applications in a single tube, is ideal for applications that require quantitation of subsets within such as cell cycle analysis, immunophenotyping and fluorescent a heterogeneous population, such as cell cycle analysis in which protein analysis different cells may be at different points of the cell cycle. Figure 1 shows an example in which flow cytometry provides information on cyclin B1 expression on a cell-by-cell basis across Compensated the cell cycle. Here, cell cycle was measured using the DNA dye DAPI and the thymidine analog ethynyl-deoxyuridine (EdU), which 5 S: 34.3% 5 10 10 is incorporated into newly synthesized DNA during the S phase. Results allow segmentation of cells in the G /G phase, S phase, 4 4 0 1 ed) 10 10 and G2/M phase. While Western blot would provide the average cyclin B1 expression level of the entire population—resulting in 3 3

10 10 a single, average value—flow cytometry allows the dissection clin B1 PE

Cy of heterogeneous cyclin B1 expression within different cell cycle 2 G2/M: 7.3% 2

10 10 compartments. The results show that cyclin B1 expression increases EdU (Eterneon R over the course of the cell cycle. 1 1

10 G /G : 57.4% 10 0 1 In this experiment, the BD FACSCelesta Blue/Violet/Red (BVR) 50 100 150 200 250 50 100 150 200 250 DNA Content (DAPI) (x1,000) DNA Content (DAPI) (x1,000) configuration—one of four available configurations to match experimental needs—was used. The three separate lasers facilitate Uncompensated the design of 3-color panels that minimize spectral overlap. This

5 S: 34.3% 5 simplifies multicolor panel design and workflow, since compensation 10 10 can be omitted without altering the biological relevance of the data.

4 4 In addition, panels composed of fluorochromes with low spectral ed) 10 10 overlap are not subject to data spread due to spillover, increasing data

3 3 quality and resolution. Such panels can be used to simplify the design 10 10 and analysis of many types of experiments, including cell cycle clin B1 PE-A

2 G /M: 7.3% 2

2 Cy (Figure 1) and immunophenotyping (Figure 2) analyses. 10 10 EdU (Eterneon R In the Figure 1 panel, DNA content was measured with DAPI using 1 1

10 G0/G1: 57.4% 10 the violet laser, Cyclin B1 expression using the blue laser, and EdU

50 100 150 200 250 50 100 150 200 250 incorporation using the red laser. Similarly, the Figure 2 panel spreads DNA Content (DAPI) (x1,000) DNA Content (DAPI) (x1,000) three cell surface markers across the three lasers to interrogate breast cancer cell line immunophenotypes. With each fluorochrome excited Figure 1. Minimal spectral overlap panel for cell cycle analysis on the BD FACSCelesta by a different laser and cross-laser excitation accounted for, spectral MCF-7 (ATCC® HTB-22™) cells were pulsed with EdU for one hour, and then fixed, permeabilized, and stained according to the recommended assay procedure for the overlap between channels is minimal and compensation is nominal, BD Pharmingen™ 647 EdU Click Proliferation Kit. After two washes to remove the click if required at all. reaction cocktail, cells were stained with BD Pharmingen™ PE Mouse Anti-Human Cyclin B1. After another washing, cells were stained with 1 μg/mL BD Pharmingen™ DAPI Solution and acquired and analyzed on the BD FACSCelesta BVR configuration. Results: Cells were gated based on light scatter, followed by doublet discrimination (not shown). The EdU data (left plots) allowed identification of cells in different cell cycle – compartments: G0/G1-phase cells are EdU with 2N DNA content (green), S-phase cells + – are EdU (blue), and G2/M-phase cells are EdU with 4N DNA content (purple). Cyclin B1 (right plots) increased as cells progressed through the cell cycle. Data with (top) or without (bottom) compensation showed similar staining patterns and equal numbers of cells in each cell cycle compartment, confirming that compensation was unnecessary for this experiment.

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For Research Use Only. Not for use in diagnostic or therapeutic procedures. Enhanced cell cycle studies using multicolor flow cytometry

With up to 12 fluorescence detectors (plus two light scatter detectors) parameter—should researchers want to multiplex fluorescent protein available on the BD FACSCelesta, cell cycle and immunophenotyping expression as well. markers can also be multiplexed together. This can save time and By pairing innovations in instrumentation with bright new reagents, sample, while also providing a deeper understanding of the cell type the BD FACSCelesta is designed to help you extract a deeper level of interest by extracting more information from one sample. Figure of biological information from your cell types of interest. You 3 shows an experiment in which cell cycle and immunophenotyping can analyze more markers, design more complex and interesting analyses were combined in a single tube. In addition, the BB515/ experiments, and ultimately achieve greater understanding and FITC channel was left open to measure GFP expression—a sixth discovery.

MDA-MB-231 Cells MCF-7 Cells EpCAM 5 5 10 10 600 4 4 500 10 10 400 3 3 10 10 ount 300 C 2 2 CD44 APC-H7 CD44 APC-H7 200 10 10 0 0 100 0

-98 2 3 4 5 -98 2 3 4 5 2 3 4 5 -98 0 10 10 10 10 -98 0 10 10 10 10 -98 0 10 10 10 10 CD24 BV421 CD24 BV421 EpCAM PE

Figure 2. Minimal spectral overlap panel for immunophenotyping on the BD FACSCelesta MDA-MB-231 (ATCC® HTB-26™) and MCF-7 (ATCC® HTB-22™) cells were stained with BD Horizon™ BV421 Mouse Anti-Human CD24, BD Pharmingen™ APC-H7 Mouse Anti-Human CD44, and Anti-EpCAM PE (Cat. No. 347211) in BD Pharmingen™ Stain Buffer (FBS), and then acquired and analyzed on the BD FACSCelesta BVR configuration. Results: Cells were gated based on light scatter, followed by doublet discrimination. MDA-MB-231 cells (left) expressed a cancer stem cell phenotype (CD24–CD44+), while MCF-7 cells (middle) were predominantly CD24+CD44+. Gates were drawn based on unstained controls. In the right plot, MCF-7 cells (light orange, unstained; orange, stained) expressed a higher level of EpCAM than did MDA-MB-231 cells (pink, unstained; red, stained). Staining patterns with and without compensation were similar, only uncompensated data are shown.

A B C 5 5

10 S: 26.7% 10 350 300 4 4 ed) 10 10 250 3 3 200 10 10 ount C 150 G2/M: 5.6% 2 2 CD44 APC-H7 10 10 100 EdU (Eterneon R G0/G1: 66.7% 0 50 2 1 10 -10 0 0

-221 2 2 3 4 5 1 2 3 4 5 50 100 150 200 250 -221 -10 0 10 10 10 10 -21 0 10 10 10 10 10 (x1,000) DNA Content (PI) CD24 BV605 EpCAM BV421

Figure 3. Multiplexed cell cycle and immunophenotyping analysis on the BD FACSCelesta MDA-MB-231 (ATCC® HTB-26) cells were pulsed with EdU for one hour and then stained with BD Horizon™ BV605 Mouse Anti-Human CD24, BD Pharmingen™ APC-H7 Mouse Anti- Human CD44, and BD Horizon™ BV421 Mouse Anti-Human CD326 (EpCAM) in BD Pharmingen™ Stain Buffer (FBS) and BD Horizon™ Brilliant Stain Buffer. The cells were then fixed, permeabilized, and stained according to the recommended assay procedure for the BD Pharmingen™ 647 EdU Click Proliferation Kit. After washing, cells were then stained with BD Pharmingen™ Propidium Iodide Staining Solution and acquired and analyzed on the BD FACSCelesta BVR configuration. Results: Cells were gated based on light scatter, followed by doublet discrimination. A. Cell cycle analysis showed clear differentiation of G0/G1 (green), S (blue), and G2/M-phase (purple) cells as in Figure 1. B, C. MDA-MB-231 cells expressed a cancer stem cell phenotype (CD24–CD44+) and a low level of EpCAM (pink, unstained; red, stained). The gate was drawn based on FMO controls. The BB515/FITC channel was intentionally left open to facilitate GFP drop-in for fluorescent protein analysis.

Ordering information Description Cat.No. BD FACSCelesta™ Flow Cytometer, BVR Configuration 660344 BD FACSCelesta™ Flow Cytometer, BVYG Configuration 660345 BD FACSCelesta™ Flow Cytometer, BVUV Configuration 660346 BD FACSCelesta™ Flow Cytometer, BV Configuration 660343

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