immunofluorescence + Flow Cytometry analysis - (Mar/13/2009 )

Dear all,

is it possible to de-attach fixed adherent cells which were immunostained, in any way, to analyse the cells for fluorescence content?
Or is the only possibility to stain the fixed cells in solution?

Kind Regards,

Dries.

Dall on Mar 13 2009, 12:54 PM said:

I would stain the cells in solution instead of trying to detach them after staining. The only ways to detach them (that I can think of right away) are trypsin, edta or some other protease/detergent treatment and all of them will damage the cell wall.....

lotus on Mar 14 2009, 03:34 AM said:

I suggest you to stain after you have detached them. and not go stain surface marker after fix(you get false negative) fix them after stained.

Thanks already for the replies!

I agree to stain first for surface markers prior to fixing, but the target proteins are intracellular, so fixation and permeabilisation is necessary for this application.

Kind regards,

Dries.

you can always spin/spot the cells again after fixation I quess?

memo on Mar 17 2009, 03:45 PM said:

I see.. with a Cytospin. Interesting..

But do these cells remain attached tight enough to withstand the several washing steps?

Greetz,

Dries.

hi,
this is an interesting link to Nunc Upcell Surface plates to obtain cells without trypsin use