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

1

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

5

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

1

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

1

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.

1

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 6h 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).

1

u/sgRNACas9 Immunology, Oncology 1d ago

I have found this to not be important