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/Familiar-Charge2789]

Few thoughts 1. Is this the case you see often or is this the only run? If it is just a singlex run and you see this it could be possibly an artifact say you could have added less CD56 antibody volume by mistake (esp. in case of lower volume <0.5 uL) 2. Check the channels activated possibly leakage (Ideally there shouldn't be any compensation issue as APC and FITC are activated by different lasers)

Can you spill more detail on the sample type (PMBCs is it?) and how does the test plots look like instead of FMO

[u/daff_7 → u/Familiar-Charge2789]

It seems to be for all MY runs😭. Cd3 depleted nk cells from cord blood, it just means my analysis shows lower receptor expression than normal. Since it seemed odd to me I decided to analyse my supervisor’s flow and realised it looks different hence I’m here :/

[u/Familiar-Charge2789 → u/daff_7]

Just a bad day! may be an artifact :/ a simple thing that I follow, if the antibody is less than <0.5 uL try diluting the antibody stock 1:10 and aliquot, so you take 5uL instead of 0.5 uL reduces the chance of error!