I have a question maybe looks naive.
I want to stain my cells with fixable viability stains. If we look at the chemistry of these stains, even live cells, although dim, can be stained.
For non-fixable viability stains, unstained cells are regularly used for gating live cells. But how about fixable live staining? Because in any case cells will be stained and if we gate them based on unstained cells, we are going to exclude some live cells.
Does anyone have any idea about it? Or is it ok to continue to gate with unstained cells?
Bests,
When using fixable viability stains, it is true that even live cells can be stained to some extent. This can make it challenging to gate live cells based on unstained cells alone, as you mentioned. However, there are approaches you can take to address this issue.
One option is to use compensation controls. Compensation is a process used in flow cytometry to correct for spectral overlap between fluorochromes. By using compensation controls, you can account for the staining of live cells and adjust your gating strategy accordingly. Compensation controls involve staining cells with single-color samples for each fluorochrome used in your experiment. These single-color controls help determine the spectral overlap between the different fluorochromes, allowing you to set appropriate compensation values.
In the case of fixable viability stains, you can stain your cells with the viability dye and then divide the stained cells into two groups: one group is used for compensation controls, and the other group is used for experimental samples. By using compensation controls, you can accurately determine the staining pattern of live cells and set appropriate gates based on the compensated data.
Using unstained cells is probably not a good idea for fixable viability dyes since, as you said, there is usually some degree of background staining on live cells.
I would recommend generating some dead cells, staining them with the viability dye and setting the gate based on that. In other words set the gate below the dead cell population. You can also kill some cells, mix them with freshly gathered (i.e. presumably alive) cells in a 1:1 ration, then stain that, so you should have 2 clear live and dead populations that you can extrapolate to your actual samples.
I do this by heating some cells in media or PBS for ~5 minutes at 65 C. Then I proceed with washing, viability dye staining and fixing (the fixing may be a bit redundant but it keeps them consistent with the other samples).
If you have a cytotoxic compound handy (like staurosporine) this could also be used.
I have attached an example of viability gates, in panel C you see the analysis of cells killed by a toxic compound, then stained with the dye.
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