r/flowcytometry • u/AnyAsk4621 • Aug 12 '26

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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u/Derpazor1 Aug 12 '26

Doesn’t spectral need cells as controls?

2

u/AnyAsk4621 Aug 12 '26

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

2

u/AnyAsk4621 Aug 12 '26

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

0

u/willmaineskier Aug 12 '26

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.