Bench Tip Video: Simplifying Cell Proliferation Studies with Flow Cytometry

 

Simplifying Cell Proliferation Studies with Flow Cytometry

Make your analysis easier with an integrated software solution

Cell proliferation analysis is used to monitor the growth rate of a cell population in response to compound toxicity, drugs and other stimulations. Cell proliferation assays using flow cytometry are becoming quicker and easier with advances in technologies and software that integrates sample acquisition and analysis.

Flow cytometry is ideal for generational analysis of cell proliferation where the number of cell divisions is tracked by dilution of a fluorescent dye. These fluorescent dyes, such as CFSE, permanently incorporate into cellular components. When a CFSE-labeled cell divides, its progeny gets half the amount of the dye and therefore each cell division can be assessed by measuring the corresponding decrease in cell fluorescence using Flow cytometry.  The more the cells divide, the less florescent the daughter cells become.

A common flow cytometry proliferation assay uses peripheral blood mononuclear cells, or PBMCs, activated with anti-CD3 and anti-CD28 antibodies or other stimulants. Although PBMCs typically have a time point collection of 4-5 days; It is important to empirically determine you specific assay endpoint in order to resolve as many divisions as possible. Using a flow cytometer with an autosampler makes it easier to analyze multiple samples for assay optimization as well as reagent concentrations. This assay can be valuable to investigate the cell proliferation behavior of different T cell populations By coupling cell proliferation analysis with cell surface marker staining, you can obtain cell proliferation information of multiple cell populations within a heterogeneous sample in a single functional assay. Here we demonstrate how to examine cell proliferation of CD4 and CD8 T cell populations in a PBMC sample.

New software technologies allow simultaneous acquisition and analysis. While the samples are running, we can begin multiparameter analysis on acquired samples. Once the first sample has been read, we can continue its analysis while subsequent samples are run. After gating the cell populations to be analyzed, a plot can be generated to obtain the cell proliferation data for these cells.

Traditional methods of cell proliferation analysis measures the total percentage of dividing cells but do not monitor distinct generations of proliferating cells. Using analysis that is integrated into the software, you can obtain more information about your proliferating cells. Flow cytometry software such as NovoExpress has a cell proliferation module that can automatically set gating of each cell division peak and generate important statistics such as the division and proliferation indexes. The division index calculates the average number of cell divisions that a cell in the total cell population has undergone. The proliferation index calculates the total number of divisions divided by the number of responding cells to understand how fast cells are growing in the proliferating cell population.

Absolute cell counts can provide valuable data on cell population growth by determining the total increase in cell number during the course of the experiment.  Some cytometers such as the Novocyte Quanteon which uses a high precision syringe driven pump for sampling can accurately determine the absolute cell count of your sample without the need of reference beads.

Once the initial sample analysis is complete, the dragging and dropping feature makes it easy to apply the same analysis to the rest of the samples in the experiment.

You can run multiple samples for each experiment following these steps, obtaining higher quality data without the need to export run reads to external software for analysis.