I have cells with strong green and red/far-red autofluorescence. I need to culture them but I cannot find a good live/dead stain. Trypan Blue reacts with salts and proteins forming clumps.
Red and green stains signals are mixed with autofluorescence.. a blue stain stands out well but Hoechst also stains dead intact cells (which I want to exclude from the live count) and DAPI is damaging/leathal.
I thought of assessing whether cell death occurs from necrosis or apoptosis but the kit I found is based on green fluorescence (which I cannot use because of autofluorescence).
Any suggestions?
If you can use flow cytometry, you can try fixable live dead dyes. Biolegend, BD Bioscience, and eBioscience have multiple types with numerous colors.
And annexin V, I guess, can also have different colors.
Thank you for you reply. I was looking for something more simple than FC (which I would need to go to another lab to perform). I will look into the suppliers you mention. The annexin V I have seen is green but maybe I should look into it more.
How bad is your autofluorescence in far red? TOPRO3 is a robust far red nucleus stain - I cannot remember if it stains live AND dead cells, but it definitely stains dead/fixed cells. I have issues with red/green autofluorescence in hepatocytes, but far red is better.
Are there any autofluorescence quenchers that work on live cells? I've had huge success with TrueBlack on my hepatocytes' red/green problem, but only ever tried in fixed samples.
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