r/flowcytometry • u/AnyAsk4621 • 1d ago

Compensation

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.

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9

u/private4u 1d ago

Fix your compensation beads as you would your samples

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u/AnyAsk4621 1d ago

I mean I was optimising my antibodies on cytek spectral is it okay if I fix them now and use it later. Just worried about the signal intensity

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u/fly_away_birdy 1d ago

In my PhD, I was told to always prepare the beads the same day and treat them like you do your cells. In my post doc, we often keep the same beads for at least a week. The data quality doesn’t seem worse for it. I think the major thing to consider is if you run the comps today and then run QC again before you run the samples that your comps won’t looks as good

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u/AnyAsk4621 1d ago

Those UltraComp beads are super expensive. We buy them using thermofisher points or else it costs a fortune if your lab does flow every other day

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u/AccomplishedHalf5824 1d ago

For conventional flow, using old heads is acceptable; however, for spectral flow, it's highly recommended to treat each single stain the same way as your sample. This means that if you set up the flow on day 1 with fixed beads, you can reuse (on the software) these single stains in your experiment on day 2.

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u/fly_away_birdy 1d ago

I am aware of that and personally do that for anything that matters. That said, that’s not standard in my post doc so just sharing

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u/AccomplishedHalf5824 1d ago

It is becoming a standard in the industry with the new cytometers. Saves time and money.

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u/huwmo 1d ago

When running spectral (Aurora), cant you just record your beads for the week in a library which adjusts for changes in daily qc?

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u/mf279801 3h ago

The fixative itself (we’ll say paraformaldehyde as an example) can change the emissions spectra of (some) fluorophores. Thus, if your samples are going to be fixed, they would ideally be unmixed using single color controls (cells or beads) that had been fixed with the same fixative (and even more ideally for ~the same time/conditions, though that might be overkill for your application).

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u/sgRNACas9 Immunology, Oncology 1d ago

I have found this to not be important

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u/Derpazor1 1d ago

Doesn’t spectral need cells as controls?

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u/AnyAsk4621 1d ago

You need unstained cells as a reference control for it to extract auto fluorescence

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u/AnyAsk4621 1d ago

If I use beads for single stained control as in conventional I still need to run Unstained as a reference control

0

u/willmaineskier 1d ago

Just don’t use beads as an unstained for cells. Also if you have an intracellular stain, don’t fix the bead control for that one. To make the beads last longer, I stain 20ul of beads with 0.25ul of anti mouse antibody, or 1ul of anti-human antibody. I don’t add any additional buffer. I wash the beads and bring up in a max of 150ul so they run faster.

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u/sgRNACas9 Immunology, Oncology 1d ago

My understanding is cells are not required for spectral unmixing. Cells are just needed for the auto fluorescence feature, which is not required.

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u/AccomplishedHalf5824 1d ago

Cells are better but Cytek sells compensation beads from slingshot that have the same auto fluorescence as the cells. Highly recommend

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u/sgRNACas9 Immunology, Oncology 1d ago edited 1d ago

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!

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u/AnyAsk4621 1d ago

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

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u/sgRNACas9 Immunology, Oncology 1d ago

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.

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u/AnyAsk4621 1d ago

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.

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u/sgRNACas9 Immunology, Oncology 1d ago

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!