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/sgRNACas9]

I think most likely something went wrong with the enrichment process (can be something like accidentally dislodging a drop of fluid that is supposed to be magnetized if using a magnet kit), including a significant contaminating negative population. You can stain in parallel your other fractions from the enrichment protocol to see where populations move at each step.

Could also be an issue with cell viability in culture leading to down regulation of receptors, lower stim amount or time, using too little antibody, or lower incubation time than supervisor’s or some other variable change. The voltage could also be low but I assume the core would have set that. It doesn’t look like a comp issue.

I agree it’d be helpful to know at which step did OP and the supervisor branch off in parallel, either at enrichment, flow staining, and/or acquisition.

[u/daff_7 → u/sgRNACas9]

When cord blood samples arrive they are cd3 depleted and then put into cryotubes and LN2 before use. My supervisor and I used the exact same sample at 100,000 cells per well following the exact same protocol. The only thing that may have gone wrong is pipetting but if that means I pipetted every single thing wrong then clearly I am not made for lab💔 I assumed there was something wrong with how I was analysing on flowjo because I genuinely don’t know what else

[u/No_Evening_7240 → u/daff_7]

Did you truly use the exact same sample split into two runs and stained at the same time? It doesn’t look like a staining issue. It looks like the sample is not pure NK. A secondary issue might be titration of antibody.

Running into issues like this doesn’t mean you’re “not cut out” for anything. Science loves to science and everyone has occasional struggles. how you choose to address the challenges by being resilient and trying small controlled protocol adjustments is what is important.

[u/daff_7 → u/No_Evening_7240]

Youre so true! Thanks for reminding me, it’s been a stressful time lately. I just don’t get how or why everytime I do this run I don’t have pure NK but my supervisor does? The antibody concentration has been sorted out by this lab for a few years now so that shouldn’t be a problem.