Cell proliferation analysis is used to support investigations into immunophenotyping, compound toxicity, drug effects on tumor cell growth, and T cell behavior under different conditions. Assays that measure cell proliferation in individual cells monitor changes in intracellular markers and/or DNA. Cell proliferation studies in cell populations report cell numbers (total and live) or measure the number of cell divisions. These assays are commonly used to study T cells and the immune response.
Flow cytometry is ideal for measuring T cell proliferation and studying unique cell types within a heterogeneous population. A selection of fluorescent dyes can be used in flow cytometry experiments such as carboxyfluorescein diacetate succinimidyl ester (CFSE) or PKH26 that incorporate into the cell membranes and intracellular structures of living cells. A decrease in fluorescence occurs as dyes are evenly diluted into daughter cells with subsequent cell divisions measured as distinct peaks during analysis. Each peak represents a successive generation of cell division. Advances in cytometer technology and integrated software have improved data collection and decreased hands-on time, but more importantly enable better quality and more in-depth information to be gathered for each sample.
The following five tips using a cytometer like the NovoCyte QuanteonTM will help you optimize your cell proliferation experiment, get the most out of your samples, and collect more information from each run.
Taking time to set up an experiment and select appropriate time points for cell collection will help you detect as many divisions as possible. The more divisions that can be resolved allows you to more accurately determine how many times each cell has divided. By optimizing dye retention and antibody titration, you are able to get more precise information on the proliferation state of the cells.
When using peripheral blood mononuclear cells (PBMCs), which typically have a time point collection range of 4-5 days, it is best to empirically test your cells for optimized cell detection. Your experiment may benefit from collecting data at more than one time point as well as titrating down the activating compound, thus allowing visualization of the number of proliferative states. Including a cell surface marker antibody stain can add phenotypic characterization of your sample, expanding the information obtained from a single functional assay.
For optimal dye retention, you may need to adjust the dye concentration for your specific cell type. Higher concentration of dye results in brighter cells and the ability to observe more divisions but also can decrease cellular function and viability. To account for this, use a labeling solution between 1-10 uM for 1-20 million cells/mL and optimize incubation time, temperature, and removal of excess dye. A typical CFSE stain with PBMCs involves a 5 uM stain in a solution containing protein (FBS or BSA) for 5 minutes at room temperature before rinsing. Since each peak represents one division, remember that 6-8 peaks are optimal for thorough analysis. It is important to ensure that the dye is not causing cell death either immediately or after long term culture.
Figure 1. PBMC Cell Proliferation analyzed on NovoExpress. PBMCs were cultured for 5 days in a 48-well plate pre-coated with αCD3 antibody (5 µg/mL) as well as αCD28 antibody (2 µg/mL). PBMCs were stained with 1 µM CFSE before culture.
In order to activate T cells and induce cell proliferation, it is necessary to add CD3/CD28 antibody or beads coated with these antibodies, or another T cell activating compound such as PHA. Correct titration will allow you to optimize the rate of cellular proliferation thus allowing you to differentiate between divisions and identify key differences between populations. Too little proliferation will not allow you to see any cell divisions while too much will decrease the capability to resolve the divisions. You can determine a saturating concentration by running a titration curve using halving dilutions of activating antibody or compound. Remember to include an unstimulated control that does not have any activating antibody/compound to determine the amount of CFSE in cells that have not divided.
Given that proliferation assays are typically limited by sample run time, having an automated plate loader on your flow cytometer can reduce time spent, increase sample numbers, and enable testing of different sample sizes and dilutions of activating compounds within one plate.
Proliferation assays typically focus on three different approaches for analysis:
Integrated software such as NovoExpress® can do quick and thorough automatic analysis using all three approaches, providing more information per experiment in conjunction with acquisition. At first glance, manually gating each division and calculating the division or proliferation index seems easy to do. However, it is difficult to set gates to avoid peak overlap and cells become over-represented after the second division round by traditional gating. Data modeling can determine the true percentage of dividing cells by correcting the frequency of each generation upon dividing and account for overlap between peaks.
Including the division and proliferation indexes provides more information on how well cells are growing within a population. The division index tells you the average number of divisions of each cell revealing how proliferative cells are in comparison to each other and gives a good idea of cell proliferation of all cells. The proliferation index indicates the number of divisions completed on average per cell that is proliferating and can reflect the kinetics of cell behavior under different conditions and samples.
Multiparameter analysis provides the ability to analyze several parameters within one experiment for more data per sample. Using instruments with increased capabilities such as multiple lasers and detectors supports multiparameter analysis and allows you to distinguish multiple types of cells in one sample, thus obtaining additional information without increasing time spent.
Absolute cell counting can enhance cell proliferation analysis by obtaining the total increase in cell number over the course of an experiment, which better determines how well cells are growing. Using an instrument with cell counting capabilities offers a look at total number of cells—in addition to looking at number of divisions—for more information per run.
An instrument with a syringe driven pump for sampling, such as the NovoCyte Quanteon, determines absolute cell count for every run without fluidic fluctuations throughout the course of the sample run. While many cytometers cannot determine the absolute cell count without the use of reference beads, direct cell count is available on these newer models.
Cell proliferation assays are incredibly useful for evaluating cell behavior and health, using these recommendations you can optimize your experiments and add valuable information, so you can get more from each experiment.
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