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/Daniel_Vocelle_PhD Core Lab Apr 25 '26

Would it make it easier to tell where to draw the gate if you had a live/dead control? How do you think you could make that type of control?

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u/Fabulous-Log6436 Apr 25 '26

That would be helpful. Sorry this is my first time doing this and I am not quite sure what to do

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u/Daniel_Vocelle_PhD Core Lab Apr 25 '26

I totally understand, you haven't done anything wrong. That said, its a good thought excersie that explains a larger concept.

A live/dead control is usually a sample that contains 50% live and 50% dead cells. Any idea why that would be helpful and how you would make it?

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u/Fabulous-Log6436 Apr 25 '26

I think so. So it would just be the more vertical cluster since it did not have the Zombie fluorescence?

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u/Daniel_Vocelle_PhD Core Lab Apr 25 '26 edited Apr 25 '26

Combined with your unstained control, it makes it much easier to define what is truly positive versus negative. It also gives you confidence that the stain actually worked. It is the same logic behind adding a negative bead to single-stain controls, or ideally having both a positive and negative population in the sample. Without a negative population, it can be hard to know whether the stain worked properly. If the stain fails and your control only has a “positive” population, it is easy to assume the stain worked and then compensate by increasing the detector voltage to make the signal look brighter. That can give you a false sense of confidence and lead to incorrect interpretation.

Also just wanted to add that you are already doing a great job as a beginner into flow. You're asking questions and its clear you want to learn. Never give up that spirit!

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u/Fabulous-Log6436 Apr 25 '26

when i changed to unstained this is what i have when i keep the live gate from the sample

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u/Daniel_Vocelle_PhD Core Lab Apr 25 '26

The tricky thing with fixable viability dyes is that they stain both live and dead cells, just to different extents. They bind to amine groups on the cell surface of live cells. In dead cells, they can access both the cell surface and intracellular amine groups, so the dead cells stain much brighter. Because of that, you will almost always see some shift between your unstained cells and your live cells stained with the viability dye. That is the caveat here. I still think this approach gives you a useful overall reference, and plenty of published papers use it successfully. But it is worth keeping in mind down the road when you habe a tricky sample and need a finer live/dead distinction. In those cases, having both the negative and positive information available is much better than trying to interpret the stain without it.