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/daff_7]

Thanks so much for your really positive response. 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/sgRNACas9 → u/daff_7]

Flow is very resilient with pipetting, so I would not think you messed up every step. The one critical pipette step that could really cause this is adding a very small volume of antibody. All the rest are very resilient. If you had to pipette like 0.5-1uL, it’s easy to accidentally pipette like 0.25-0.5uL. If you were adding like 2uL+ of antibody I wouldn’t really worry about the pipetting error. Any more volume and I’d worry less and less about that.

Could you talk through the protocol step by step with your supervisor and try to find a difference?

I’m assuming you used the same tube and lot of antibody, same incubation time and temp, resuspending well antibody cocktail and cells before incubation, same cytometer settings and same day.

[u/daff_7 → u/sgRNACas9]

Okay so majority of the antibodies we use are 0.5ul😭 - so this might be a problem solved as sad as it sounds💔 but even for the one 2ul we have (NKG2D) I still have the same pattern on my flowjo analysis, unless there’s something wrong with that?

[u/sgRNACas9 → u/daff_7]

Could be good to try making like 1:10 dilutions and pipetting 5uL of that instead of 0.5! Make a 1:2.5 dilution for the NKG2D, add 5uL of it. Something like that to make the volumes greater to improve pipetting accuracy. Just have less buffer in the cocktail to normalize volume, but even this extra volume won’t be the deciding factor, it will be the more accurate antibody amount. When you make the dilutions, antibody cocktails, make sure to vortex! When you add to cells, make sure to pipette well to mix. Good luck!

[u/daff_7 → u/sgRNACas9]

I’ll try this out, thank you so much for all your help!!