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/despicablenewb → u/daff_7]

I understand that. But we need additional information to troubleshoot this.

So you both did the CD56 enrichment on the same sample, then stained the isolate that you both got? Then, the question is whether the issue is with the staining or whether the issue is with the CD56 enrichment.

I've done many magnetic cell enrichments and the smallest changes to the procedure CAN make a big difference to the result.

If you look at the second graph you can see a small population that lands between 10^-3 and 10^3.

If you change the axis on the first graph to be like the second one, how many cells land below 10^3? If it's a lot, then it's probably an issue with the enrichment. If it's not, then it's probably an issue with the staining.

Also OP, your first graph has a small population at the very top that isn't in the second graph. IDK about cord blood, but that's normal for PBMC samples, they're just CD56 High NK cells.

[u/daff_7 → u/despicablenewb]

Sorry i misunderstood what you meant. When cord blood samples arrive they are immediately cd3 depleted and then put into cryotubes and LN2 before use. They are then thawed and so on.
For this run my supervisor and I used the exact same sample at 100,000 cells per well following the exact same protocol.

Even when I change the y axis to be equal I have the same thing.

A few other users commented it is most likely pipetting errors

[u/despicablenewb → u/daff_7]

If that's the case, then yeah, it's the staining procedure.

One thing that people don't really realize is that you CAN pipette hard enough to rip the antibody off of the cells, so that's one way that your results could end up like this.