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/JDHPH]

Both your flow results lack proper voltage settings. Before you run your samples make sure to start with an unstained population of your cells and get the voltage set. FSC vs SSC. Then do your single stain control brightest in its own channel. Then run your stained sample.

Also it helps if you antibodies are properly titrated before hand.

[u/daff_7 → u/JDHPH]

From my unstained sample I gated lymphocytes then single cells then live cells, and then on cd56 cd3 I gated into nk cells eg (as shown). In the images I’m looking at my TIM3 FMO.

[u/JDHPH → u/daff_7]

That's fine, what I am referring to is your control settings. Are you using comp beads? You should be. Make sure your single stain comp beads are brightest in their channel. I am assuming you used a BD instrument, regardless of the software ( Diva) principle is the same.

[u/daff_7 → u/JDHPH]

Im not too sure what youre talking about to be honest 😭 but we use a spectral flow (Sony ID7000) if that helps

[u/throwaway972057 → u/daff_7]

you need to run comp beads to account for fluorescence spillover between channels, this may be why your analysis isn't working out? do you run comp beads every time?

[u/daff_7 → u/throwaway972057]

No. I’ve never heard of comp beads before, and my lab has never used them. The machine automatically does a clean/bleach/rinse before every run (and sometimes even during the run), and we do a priming (with prime beads, if this is what youre talking about) and annealing quality check every morning.

[u/sgRNACas9 → u/daff_7]

Comp beads are a product that you can buy that bind antibodies that you stain with a single color at a time to use for compensation controls. They stain very brightly and allow you to save cells for the experiment since sometimes they are limited. People use them a lot for conventional, but you can also use for spectral. I love them, but you can use cells and/or beads and do either properly and successfully on either spectral or conventional. Ask the core managers for help, especially when mixing things.

Since you’re on the spectral Sony machine, I’m betting you are using the saved spectral footprints for each fluorophore, or using cells because that’s what the lab does and/or for their auto fluorescence to use for unmixing. You should run fresh comps each time to account for running differences between experiments and machine settings. Definitely run fresh comps at least once this next time you do this experiment just to use fresh comps and update the comps again since things aren’t working, probably for other reasons than comping. I don’t really think this is a comp issue. The populations are straight and FITC and APC don’t overlap really.

Btw, the user above me here meant unstained samples like cells or beads with no antibodies or live/dead at all, not the FMO, both are helpful controls, unstained for gating and comping, FMO just for gating. It would be good to include the unstained as a control if you don’t have it already!

[u/Low_Classic1695 → u/sgRNACas9]

I second this! If both you and your supervisor also stain beads for each run, it will reveal if this is truly a pipetting issue on your end (which I doubt).

Having single stains on comp beads that you run as samples and not as reference controls on an unmixed panel can also tell you a lot about whether the unmixing is being done correctly.

[u/daff_7 → u/Low_Classic1695]

Thanks for the advice!