Hi everyone,

I guess this is a basic immunostaining question. I am preparing cells for flow cytometry. First I'm staining for a pluripotency marker, then I'm staining for BrdU.

I add 100 uL of SM (2%FBS, NaN3 in 1xHBSS), then 80% EtOH and incubate at 4*C for 30 minutes or more. Then I permeabilize with BD cytowash/perm buffer (a saponin based buffer used to permeabilize cells) and then I incubate O/N with an antibody against the pluripotency marker. When I retreive the cells in the morning, the cells become one giant mass that cannot be dissociated into single cells. I can no longer proceed with the rest of my staining.

Does anyone know what could be happening? I would love to know peoples thoughts on this

Thank-you in advance!

Can you try immunostaining with antibody for just an hour rather than overnight? If you have to do it overnight, can you leave out the permeabilization (maybe using a methanol fix 5min at -20 degrees to both fix and permeabilize)? I assume that not poking holes in the cells will keep them happier for a longer incubation.
What pluripotency marker are you using? If it is on the surface of the cells, you could leave out permeabilization, maybe try another fix like paraformaldehyde.

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It might be better try to dissociate the cells to single cells, then perform fix, permeabalization, and stain. All the procedure can be performed in a FACS tube for washing and changing the solutions.

It is impossible. Probably EtOH is not cold (at least -20C)

Hi Dmitry,


Could you please explain why it is impossible? I have been using this method for staining intracellular antigen for suspened cells.

Hi !

I have misunderstood:)

You are rigth. I meant that it is impossible to destroy aggregates after fixing. and what they arise during staining (warm or old reagents).