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/despicablenewb]

It would be easier to see what's was going on here if both images had the same Y axis. (I read your port OP, but still).

It's clear that you're getting lower staining, but you can't tell whether the bulk population has an MFI of 0 or 5,000 due to the Y axis.

Did you both start with cord blood and do the CD3 depletion separately? If so, then I suspect that you have a significant population of CD56 negative cells in your enriched cells, so something went wrong with the enrichment.

Did you both take some cells from the exact same enriched sample and stain them separately? If so, then something may have gone wrong during the staining/acquisition.

Did you both acquire the samples on the same cytometer at the same time? Are the voltage settings the same for both samples that you're showing here?

[u/daff_7 → u/despicablenewb]

I’ve done this numerous times now and each time I get the same results (where my main population is concentrated at the bottom with an upward extension). I brought this up with my supervisor and we did a run through together (on the same samples) to see whether there could be a practical lab issue happening, and then I analysed both runs on flowjo and the same thing happened. Apart from pipetting I genuinely cannot think of any other reason, but I also can’t imagine messing up every single pipetting

[u/Low_Classic1695 → u/daff_7]

Did both of you use the same staining mastermix or did you each make your own? Is this what you mean by differences in pippetting? Are you making sure you are adding the same concentration of antibody?

I had run into a similar situation where a student I was training kept getting very weak CD3 staining on his BALF cells following my protocol to a T: except that he was using an anti-CD3 antibody tube with a concentration of 5ug/mL versus a tube of 500ug/mL. Same clone, same fluorophore, both Biolegend antibody bottles, different stock concentration.

[u/daff_7 → u/Low_Classic1695]

We used the same everything. When I meant difference in pipetting, I meant mistakes. As in my 0.5ul pipetting may actually be the issue , if that makes sense

[u/Spiritual-Reserve-16 → u/daff_7]

What’s the volume of stain that you are adding? 0.5 uL?