Immunostaining for flow cytometry - (Jun/24/2009 )

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!

Have you tried vortexing the samples while you add the ethanol? When I fix cells, I always gently vortex (maybe around 4 or 5) while I slowly add the ethanol using a P1000. Give it a try. Hope this helps!