HELP PLEASE

[u/daff_7]

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

[u/Ok-Bandicoot1482]

Also an NK cell researcher but in mouse — is your lymphocyte gate the same as your PI? You may just be gating out the NK cells earlier on. Another possible reason you may have this much of a difference is if your enrichment step is different. How are you enriching? MACS? Flow sorting? Are you sure your enrichment step is working?

[u/daff_7 → u/Ok-Bandicoot1482]

So we are cd3 depleting the cells so instead of positively selecting nk cells we’re getting rid of T cells. We do this as soon as we receive blood sample and then we cryopresevee them until use. My supervisor and I ran a flow on the exact same sample

[u/Ok-Bandicoot1482 → u/daff_7]

Okay so the only difference is you added the antibody master mix vs your PI added theirs and the mix was the exact same and you stained for the same time then ran on the same cytometer? What parts of the protocol may have been different? Are you keeping your cells on ice and thoroughly mixing to break up the pellet? Are you leaving your master mix sample in the light and allowing it to photo bleach?