Hello,
I am measuring multiple different parameters in cells using fluorescent channels in flow. I would also like to assess well viability. Given that I take the same volume from each well, and do not define a stopping event, can I compare live events between wells as a viability measure? I understand that not all cells that appear live in flow are viable, thus the use of viability stains. Do such stains have to be used to generate viability data from flow?
Thanks!
Hi David,
The short answer is that the use of a viability dye is required. Even with taking the same volume from each well, without a viability dye when you compare data the number of "live cells" in the gate could be different for many reasons other than non-viable cells (changes to adhesion, cellular proliferation, etc.). I would recommend using a viability dye or cell death kit (ApoTracker / Annexin V) to attain the information you are looking for. Our lab uses the following dyes extensively for our flow analysis:
https://www.thermofisher.com/order/catalog/product/L10119 (1:1000 dilution after resuspension in 50ul DMSO)
thermofisher.com/order/catalog/product/L34966?SID=srch-srp-L34966 (1:100 dilution after resuspension in 50ul DMSO)
Alternatively, some labs use Propidium Iodide or DAPI to determine viability since these dyes cannot enter live non-fixed cells.
Hope this helps
Hi David,
You can do this if you express the viability in %.
Hope this helps
Yes, cell viability can be assessed with the Live Gate in flow cytometry. The Live Gate includes two parameters: forward scatter (FSC) and side scatter (SSC). Cells with higher FSC and SSC values are typically considered more viable than cells with lower FSC and SSC values. Therefore, the Live Gate can be used to identify and measure the viability of cells in a sample.
In flow cytometry, the live gate is typically used to distinguish live cells from dead cells or debris based on their forward scatter (FSC) and side scatter (SSC) properties. While the live gate can provide a quick assessment of the population of cells that appear to be alive, it does not directly measure cell viability.
Cell viability refers to the functional state of cells, indicating whether they are alive and capable of normal cellular activities. To accurately assess cell viability, additional measures are required, typically involving the use of viability stains or dyes. Viability stains such as propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) are often used to distinguish between live and dead cells based on their ability to exclude or penetrate these dyes.
The use of viability stains is important because not all cells that appear live in flow cytometry are necessarily viable. Some cells may have compromised membrane integrity or other functional impairments, which cannot be determined solely based on their forward scatter and side scatter properties. Viability stains provide a more accurate assessment of cell viability by specifically labeling non-viable cells.
Therefore, if you want to generate reliable viability data from flow cytometry, it is recommended to use viability stains in addition to the live gate. This approach ensures that you are accurately distinguishing between live and dead cells, providing a more comprehensive assessment of cell viability in your experiments.
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