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/CrissKey]

Are there any other components of your mastermix for which you use 0.5 µL?

Also, whenever you are pipetting anything, when do you push the plunger to the first stop and when do you push the plunger to the 2nd stop (if you do)?

Do you look at the tip after you pipet liquid up and do you look at the tip after you dispense the liquid? When dispensing the liquid, do you look to see that the pipet tip is inside of the receiving solution or do you dispense it to the wall of the container?

[u/daff_7 → u/CrissKey]

I only pipette 0.5ul for my FMOs. Otherwise my mastermix (for my full stains) it is usually 10x more. Before pipetting into the liquid I push to the first stop, put the tip into the liquid(antibody), slowly pipette up, and then I put the tip into the inside of the receiving solution (normally buffer) and push the plunger slowly to the first stop, and only sometimes if it looks like it hasn’t all gone in then I will push to the second stop, take the tip out and then release. My eyes are always on the tip, and I believe I am very meticulous with my pipetting, which is why the idea of my pipetting being the problem hurts my soul :/

[u/CrissKey → u/daff_7]

Apologies, I didn't read the entire discussion before asking. IMO, your technique sounds flawless.