u/cd244

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!

Hi guys,

I'd like to hear your thoughts on staining cells in 96-well plates vs. FACS tubes.

I've always stained cells in 96-well plates because I usually have many samples at a time. My protocol is pretty straightforward:

• Add 1 million cells per well.
• Centrifuge and discard the supernatant.
• Resuspend the cells in 50 µL of antibody mix.
• After staining, wash with 200 µL FACS buffer, centrifuge, and discard the supernatant. Repeat 3 times.
• Fix the cells in 50 µL PFA.
• Centrifuge, discard the supernatant, and wash 3 more times with 200 µL FACS buffer.
• Finally, resuspend in 200 µL FACS buffer for acquisition.

I'm about to process around 400 samples, but one of my colleagues insists that I should stain everything in FACS tubes instead.

To me, using tubes seems much slower since we can only process a few samples at a time. I also feel like we'd lose more cells when pouring off the supernatant after centrifugation.

For those of you who stain in FACS tubes, what's your experience? How do you remove the supernatant—by pipetting it off or by simply decanting (inverting) the tubes? Have you noticed any significant cell loss?

Thanks!

Hello, I would like to know does anybody have a protocol for using BD brilliant staining buffer since their protocol is really unclear : https://www.bdbiosciences.com/en-eu/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/brilliant-stain-buffer.563794?tab=product_details

We are staining human PBMC in 96 well plates, we use 50 ul FACs buffer per well with cocktail of diluted antibodies. BD recommend to add 50 ul brilliant staining buffer per well regardless of the volume of cocktail. Does that mean that I have to stain my cells in 100 ul of volume and have to double my antibodies? Does anybody have stained cells directly in brilliant staining buffer ?