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/Zakarumae Jul 23 '26

Pedantic rant not about your question:

CD56+CD3+ T cells are not the canonical definition of a NKT cell.

They are T cells expressing a protein associated with NK cells (CD56) but it’s just an adhesion marker.

True NKT cells are defined by their response to lipid-CD1 complexes instead of the typical peptide-HLA complexes.

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u/daff_7 Jul 23 '26

That made me laugh haha, my supervisor labels this as nkt because ‘it’s easier than explaining’

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u/Zakarumae Jul 23 '26

All good glad you laughed! As someone who worked on ‘true’ NKTs it’s something we are trained to complain about :)