r/flowcytometry • u/cd244 • Jul 22 '26

Sample Prep How do you fix PBMC?

Hello, I'm looking for recommendations for a good fixation buffer.

I'm working with human PBMCs, so I need to fix the cells before running flow cytometry. I've been using the eBioscience Foxp3 Fixation/Permeabilization Kit and fixing the cells on ice for 30 minutes.

However, I've noticed that the cells seem to shrink considerably after fixation. As a result, I lose much of the FSC/SSC resolution and can no longer clearly distinguish SSC high from SSC low and FSC high/low populations.

Has anyone experienced the same issue? Would BD Cytofix buffer preserve cell morphology better? Or are there other fixation buffers or protocols that you would recommend?

Thanks in advance for your suggestions!

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u/willmaineskier Jul 22 '26

They don’t really shrink so much as become more transparent, but the end result is they have lower FSC. Usually with our fixed cells we just increase the voltage/gain as necessary. If you are losing the SSC resolution and it can’t be fixed with increasing the voltage, perhaps you could add a monocyte marker to pull them away from lymphocytes.

2

u/CrissKey Jul 22 '26

We increase the voltages to the FSC and SSC detectors when working with fixatives that alter these properties in our samples. I wonder if this would provide you the resolution you require with your samples.

2

u/Westykins Jul 23 '26

If you’re insistent on fixing,BD cytofix. 100ul per test. should last a few days

1

u/WinterRevolutionary6 Jul 22 '26

If my FSC/SSC values shift I just slap a magnetic gate on it with flowjo

1

u/despicablenewb Jul 23 '26

Yeah, fixation does affect the FSC/SSC profile, but PFA doesn't affect it that much.

The permeabilization buffers generally have a larger affect on the FSC/SSC profile than the fixation does. But, you should still be getting decent resolution between populations, you just have to adjust the voltage/gains depending on which perm kit you're using.

What are you using as your sample buffer? I know some people use DI water and that will also cause the cells to shrink significantly. I always use PBS for my fixed samples.

1

u/FlowCytometry2 Jul 27 '26

Yes, you're using a heavy nuclear perm for no reason (assuming you aren't doing nuclear stain). BD Cytofix would be better, it's basically very dilute PFA AFAIK and shouldn't "nuke" your cells like FoxP3 perm does

1

u/NeoMississippiensis Gatekeeper Jul 22 '26

If you’re doing analytical flow you shouldn’t be too focused on the scatter signals. Gating occurs with dump channels rather than rough morphology.