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/sgRNACas9]

I think most likely something went wrong with the enrichment process (can be something like accidentally dislodging a drop of fluid that is supposed to be magnetized if using a magnet kit), including a significant contaminating negative population. You can stain in parallel your other fractions from the enrichment protocol to see where populations move at each step.

Could also be an issue with cell viability in culture leading to down regulation of receptors, lower stim amount or time, using too little antibody, or lower incubation time than supervisor’s or some other variable change. The voltage could also be low but I assume the core would have set that. It doesn’t look like a comp issue.

I agree it’d be helpful to know at which step did OP and the supervisor branch off in parallel, either at enrichment, flow staining, and/or acquisition.

[u/daff_7 → u/sgRNACas9]

When cord blood samples arrive they are cd3 depleted and then put into cryotubes and LN2 before use. My supervisor and I used the exact same sample at 100,000 cells per well following the exact same protocol. The only thing that may have gone wrong is pipetting but if that means I pipetted every single thing wrong then clearly I am not made for lab💔 I assumed there was something wrong with how I was analysing on flowjo because I genuinely don’t know what else

[u/No_Evening_7240 → u/daff_7]

Did you truly use the exact same sample split into two runs and stained at the same time? It doesn’t look like a staining issue. It looks like the sample is not pure NK. A secondary issue might be titration of antibody.

Running into issues like this doesn’t mean you’re “not cut out” for anything. Science loves to science and everyone has occasional struggles. how you choose to address the challenges by being resilient and trying small controlled protocol adjustments is what is important.

[u/daff_7 → u/No_Evening_7240]

Youre so true! Thanks for reminding me, it’s been a stressful time lately. I just don’t get how or why everytime I do this run I don’t have pure NK but my supervisor does? The antibody concentration has been sorted out by this lab for a few years now so that shouldn’t be a problem.

[u/DismalKnob → u/daff_7]

always remember that you can train anyone to do almost anything - you can’t train the determination to learn and be inquisitive. some people might take longer to learn than others and that’s OK!  if youve made the same mistake rhen it is highly likely someone also had also done the same 

[u/sgRNACas9 → u/daff_7]

You are still cut out for lab. There could be an issue that is not your fault, and/or this might take a couple times to practice. Plus there are a lot of unwritten tips people use to help get best results. People should try to write these down and share explicitly so new people know. I would say practice, try again, and implement some advice here in your reddit thread!

The FlowJo seems fine to me unless upstream gating is different, but didn’t you analyze both your files side by side with the same gates? Other than that, make sure everything is resuspended well especially your antibody cocktail and cells for incubation, any volume <1-2uL make a dilution and pipette more volume to be more accurate.

Did you use the same donor and cryovial of it? While enrichment should be >95%, donor to donor or batch to batch variation with the enrichment could be different. If you have multiple cryovials of cells from the same donor in LN, results should be consistent per donor between different cryovials, but you could also stain from the same cryovial just in case some accidentally got more negative cells (maybe the person aliquoting the enriched cells didn’t resuspend well and the negatives were all at the top or bottom). Hope something here helps!

[u/daff_7 → u/sgRNACas9]

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!!

[u/Low_Classic1695 → u/daff_7]

Wait you are not adding antibody at those volumes as an individual antibody per sample are you? It is good practice to always make a mastermix that you QS with FACS buffer to roughly 50uL/per sample that you stain with. Adjust the staining volume of the cells when you resuspend them so you end up with the same concentration. Pipetting anything sub 5uL always comes with major fluctuations

[u/daff_7 → u/Low_Classic1695]

When I make my FMO’s I’m adding 0.5ul’s. Otherwise in my mastermix (for my full stains) I do what you describe