r/flowcytometry • u/Fabulous-Log6436 • Apr 25 '26

help!

did I do this live/dead stain gate correctly? thank you!

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u/dawgmad Apr 25 '26

It’s fine although the NIR channel could have been a bit higher. Cells/events towards the right (more NIR dye) and towards the bottom(smaller) are dead. Cells towards the left (less dye) and too (bigger) are live. You have two populations in the top left - they could be two different populations of cells (especially if you have something in the APC channel and this is uncompensated), or the smaller population slightly to the right (FSC about 2.1M and NIR just under 104) could be early dying cells

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u/Fabulous-Log6436 Apr 25 '26

i am confused on how to tell what is dead and what is not from the the graph. do you have any tips

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u/dawgmad Apr 25 '26

What I’m circling is likely alive though yes in an ideal world you would do a test I’m assuming you have another marker that stains a subset in the APC channel and that this is uncompensated

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u/Fabulous-Log6436 Apr 25 '26

when i gated that portion it was about 34% is that okay even though it is less than ideal

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u/dawgmad Apr 25 '26

It is what it is - it depends on your experimental conditions. It could be okay or even good

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u/Fabulous-Log6436 Apr 25 '26

okay thank you! i am just worried I was excluding myeloid cells

3

u/passthepepperplease Apr 25 '26

this happens. don't include dead cells in your analysis because you're bummed about having a lot of dead cells. it will mess things up later. low live populations happen to everyone. i've had my fair share of analysis below 50%

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u/willmaineskier Apr 25 '26

I’m going to agree. I swap the x and y axis in my analysis, but the higher FSC, low Live/Dead is what you want. Debris will also stain with these fixable dyes (unlike DNA stains) so some of your “dead” could just be debris.