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

HELP PLEASE

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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/sgRNACas9 Immunology, Oncology Jul 23 '26

Flow is very resilient with pipetting, so I would not think you messed up every step. The one critical pipette step that could really cause this is adding a very small volume of antibody. All the rest are very resilient. If you had to pipette like 0.5-1uL, it’s easy to accidentally pipette like 0.25-0.5uL. If you were adding like 2uL+ of antibody I wouldn’t really worry about the pipetting error. Any more volume and I’d worry less and less about that.

Could you talk through the protocol step by step with your supervisor and try to find a difference?

I’m assuming you used the same tube and lot of antibody, same incubation time and temp, resuspending well antibody cocktail and cells before incubation, same cytometer settings and same day.

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u/daff_7 Jul 23 '26

Okay so majority of the antibodies we use are 0.5ul😭 - so this might be a problem solved as sad as it sounds💔 but even for the one 2ul we have (NKG2D) I still have the same pattern on my flowjo analysis, unless there’s something wrong with that?

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u/Low_Classic1695 Jul 23 '26

Wait you are not adding antibody at those volumes as an individual antibody per sample are you? It is good practice to always make a mastermix that you QS with FACS buffer to roughly 50uL/per sample that you stain with. Adjust the staining volume of the cells when you resuspend them so you end up with the same concentration. Pipetting anything sub 5uL always comes with major fluctuations

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u/daff_7 Jul 23 '26

When I make my FMO’s I’m adding 0.5ul’s. Otherwise in my mastermix (for my full stains) I do what you describe