BD FACSCelesta

Cell Analysis in a New Light

Flow cytometry can provide rich data to cell biologists working in a wide range of fields, from molecular interactions to systems biology, from pharmacokinetics to cancer biology, from cell signaling to marine biology to biophysics. Use of flow cytometry has blossomed with intensified interest in proteomics, increased use of biomarkers in drug development research and the spread of high-throughput, cell-based drug screening research.

Detecting and analyzing light scatter allows researchers to measure physical characteristics of cells in suspension, such as cell shape, size and internal complexity. Adding fluorescent markers allows researchers to interrogate expressed or secreted proteins that reveal cell phenotype, function and status. Using a broad landscape of cell function assays, flow cytometry can shed light on a variety of sample types, such as whole blood, cell lines and yeast. With three lasers and up to 14 optical channels, the BD FACSCelesta™ flow cytometer can multiplex many of these assays at once on the same sample.

Study Complex Populations at the Single-cell Level

A major strength of flow cytometry is its ability to study complex populations. Western blots, immunoprecipitation and PCR-based techniques rely on lysing the entire sample. Their results provide useful data for the population as a whole, but it can be difficult to compare subsets of cells that might behave differently. Other techniques that examine individual cells, such as microscopy, are hard to scale up for quantitative analysis. Flow cytometry can characterize large numbers of individual cells, allowing it to identify, quantify and characterize different subsets of cells in a heterogeneous population.


3-Laser BVR Configuration

Two-color flow cytometric analysis of apoptosis and viability in Jurkat cells
Two-color flow cytometric analysis of apoptosis and viability in Jurkat cells
Jurkat cells were treated with 0.025% DMSO vehicle (top plots) or 5 μM of camptothecin (bottom plots) for 4 hours, harvested from culture, washed and resuspended in Annexin V Binding Buffer. Cells were then incubated with BD Pharmingen™ APC Annexin V (Cat. No. 550475) and BD Pharmingen™ Propidium Iodide Staining Solution (Cat. No. 556463) for 15 minutes at room temperature protected from light, and then analyzed by flow cytometry on a BD FACSCelesta system. Debris was excluded based on the light scatter properties of Jurkat cells (left plots). DMSO vehicle-treated cells were primarily Annexin VPI, indicating that most cells were live. Camptothecin-treated cells show an increase in the number of Annexin V+ (apoptotic) and Annexin V+PI+ (dead) cells, indicating that camptothecin treatment induced apoptosis and cell death.
Minimal spectral overlap panel for cell cycle analysis on the BD FACSCelesta
Minimal spectral overlap panel for cell cycle analysis on the BD FACSCelesta
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 BD Pharmingen™ 647 EdU Click Proliferation Kit. After two washes to remove the click reaction cocktail, cells were stained with BD Pharmingen™ PE Mouse Anti-Human Cyclin B1. After another washing, cells were stained with 1 μg/mL of 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.
Multiplexed cell cycle and immunophenotyping analysis on the BD FACSSCelesta
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. B, C. MDA-MB-231 cells expressed a cancer stem cell phenotype (CD24CD44+) 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.

3-Laser BVYG Configuration

Expression of fluorescent proteins in transfected human embryonic kidney cells
Expression of fluorescent proteins in transfected human embryonic kidney cells
HEK-293 cells were transfected over 24 hours with AcGFP (left) or mCherry (right), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and cryopreserved for one week. After thawing, cells were washed and then analyzed by flow cytometry. Transfected cells (green or red) were compared to wild type (black) for expression of AcGFP or mCherry, respectively.