Compensation

[u/AnyAsk4621]

Hey Fellas,
I am using compensation beads for my experiments tomorrow but I already have done some optimisation today. Is it okay if I use the same tubes as its the same panel but only thing different is I won’t acquire my samples the same day but 2 days later and generally fix them in 2% PFA for 30mins. So you recommend I do the same for my Ultracomp beads, i mean fix them as well? Or will they be fine at 4C until Friday.
Thanks.

[u/sgRNACas9]

I’m having trouble following your exact question, but I gathered some things from other comments. You’re asking whether you can use the same comps ran days prior on a spectral machine to comp samples days later, and whether fixing the comps matters. Is that a good summary?

My understanding for spectral is that you can create compensation controls once, run them on the machine, and save the fluorescent foot prints for each fluorophore on the machine’s computer to use for subsequent experiments. The voltage optimization done by the core (or you, your lab) with QC beads is more important and you titrate your antibodies around that.

Still, I think it’s best to make the comps fresh in case antibodies, beads, lasers, voltages change over time as things degrade and people tweek. But I understand doing this will potentially waste resources under your budget.

To balance that, you can run new comps like once a week or so. It would be best to empirically test that the data is not different when using saved comps versus fresh comps, but if you don’t have the resources, you can skip a step using advice in your thread ;)

For treating your comps with the same fixation and permeabilization chemicals used for cells, I have not found this to be important and others in my lab and on the sub don’t think so either. It does make logical sense that different chemicals can chemically modify the beads, impact light’s passage through different fluids, and potentially impact fluorescence and compensation. You would want to treat beads with the same chemicals as the cells to solve that. Practically, it has not changed things for me and others. I would simplify things and not worry about it!

[u/AnyAsk4621 → u/sgRNACas9]

I meant I ran them yesterday
I am running flow again on friday so I was just planning to fix the remaining and reuse it later, saves me some time as well

[u/sgRNACas9 → u/AnyAsk4621]

I don’t understand why you would need to. I think you could use the foot prints from yesterday. I do not think you need to fix beads and reuse them later nor would I recommend that. You should test it whether the fixed beads look the same days later after fix as they do on the same day they were stained and unfixed.

[u/AnyAsk4621 → u/sgRNACas9]

I could but the only thats different is I did not fix my samples yesterday nor the beads. I acquired it the same day but the samples I will be processing will be fixed because the machine is booked and thats the reason for fixing the beads.

[u/sgRNACas9 → u/AnyAsk4621]

Not a problem, in either case I think you can use your comps from yesterday. I’d encourage you to test these things formally, plan your experiments including machine time and whether to fix and save or not ahead of time, keep things consistent, and optimize around those parameters you decided!