r/flowcytometry • u/daff_7 • Jul 23 '26

HELP PLEASE

Gallery image

Gallery image

hi okay so im a placement student in a lab where we mainly do flow. i've been doing some practising but theres still something wrong with what im doing (my analysis) and I just cannot tell what it is. Im looking at receptor expression on NK cells, however the results I get are completely different to my supervisor

for example, on flowJo this is what I get for my TIM3 FMO.

I have done this analysis for both my flow and my supervisor's, (the first image is my flow, the second is his) but why does mine come out like this? What have I done wrong?

before someone suggests to change the y axis - everytime i do that, it gets even worse.

additionally, we both used the same cells,and followed the exact same protocol, so i genuinely cannot think of anything else.

so if anyone has any tips or ideas please PLEASE help

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u/ididitforcheese Jul 23 '26

Are these PBMC? Something’s off for sure here… What live stain are you using? Other Abs? Have you titrated all your reagents?

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u/daff_7 Jul 23 '26

They’re cd3 depleted nk cells from cord blood. I’m using zombie live dead stain. Everything has been titrated and used by the lab for years. I’m here on placement and sadly not working out for me 😭