Hi all,
I have a naive question and would like some opinions.
I performed 2 flow cytometry runs with different samples, and I found more dead cells in my second run. My question is will the increase in dead cells affect my subsequent count for immune cells? My gating strategy is lymphocytes> single cells> live cells> CD45+ immune cells> ....
The reason I ask this question is because we also take the bead ratio into account and whether the amount of dead cells present will affect the immune cell population?
Thank you.
Hello, first of all, did you check your cells condition before harvest? even though you don't see dead cells or can't recognize them, preparing cells for flowcytometry may induce cell death especially if the protocol you are using is not optimized, so be sure of that too.
The answer to your question is obviously yes. my suggestion, is minimizing dead cells population and try to make sure that you don't include them in your final analysis of the different popultions you want to detect.
Good luck.
Hello Alex Peh
Yes, it will. The dead cells present in your sample can greatly affect your staining and therefore the quality of your data. The dead cells have greater autofluorescence than living cells and will increase non-specific antibody binding, thereby leading to false positives. This may make the identification of weakly positive samples and rare populations difficult. Moreover, cell clumping may occur due to the release of DNA from dead cells which is sticky in nature. Thus, identifying and removing data points representing dead cells is a critical step in ensuring accurate results.
There are dyes available which can distinguish live from dead cells. Depending on the fluorescent markers used, either PI, 7AAD or DAPI may be added to cells just prior to analysis. Only dead cells will take up these dyes. A very small amount of dye may be used to check viability and will not be detrimental to the cells. As little as 0.5 - 1.0 ug/ml of the dye may be added before the start of acquisition. However, if your sample is fixed, these dyes will not work. You will need a fixable live/dead marker. These nuclei acid binding dyes, when bound to double stranded nucleic acid will fluoresce. The dead cells can be identified and removed from the final analysis by gating on the unstained population (live cells).
Best.
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