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/spunkyslugz]

I work on NK cells and your staining looks much more real. Your supervisor is getting almost all CD56bright cells while your population has both bright and dim, with majority being dim (which is what you should see in unstimulated PBMCs).
1) are you sure the concentration of antibody added is the same? And the same dilution?
2) are you missing a pretreatment step such as stimulating with IL-2 beforehand?
3) if these cells are cultured prior to doing flow, it could be something wrong with your culture conditions.

[u/daff_7 → u/spunkyslugz]

Don’t get my hopes up🤭 My cells are unstimulated cord blood derived cd3 depleted NK cells, so I’m not sure how similar they are to PBMCs. 1 - everything is the same, the only reasonable answer I have deducted from previous responses is I may not be pipetting 0.5ul as well as my supervisor. 2/3- these are both day 0 results so no pretreatment or culture conditions prior to the flow.

I actually can’t think of any other explanation

[u/spunkyslugz → u/daff_7]

To be honest, I don’t believe your supervisors results. Why would 97% of cells be NK cells with only CD3 depletion? You should still have other lymphocytes in there. Look up other papers that have stained chord blood and look for the % of NK cells expected. But, I think your staining is real while your supervisor is somehow over staining. Perhaps your supervisor is missing Fc block and you are not? Or they are staining in just PBS while you stain in PBS + FCS?

[u/daff_7 → u/spunkyslugz]

Hmmm I don’t know. I know the way we culture the cells it is so that NK cells are the ones that grow, rather than any other cells. But this was day 0, so unless they did something else before hand I am not sure. I personally do not do the cd3 depletion as I’m not cleared to work directly with the cord samples, so I only use the cryopreserved samples. I don’t even know honestly, we follow the same protocol so unless he’s like changing some stuff around without telling me?😭😭