Dear Paul,

I recommend first stain the cells with surface markers, then fix them and later permeabilize, at the end stain with intracellular antibodies.

Dear David the protocol you used should work fine. just remember that if you fix and permeabilize cells you can't use standard viability dyes such as 7AAD but you have to use fixable dyes such as zombie aqua by life tech and follow their instructions for cell staining.

Dear Paul,

I recommend first stain the cells with surface markers, then fix them and later permeabilize, at the end stain with intracellular antibodies.

Dear Nicola,

My method is working. Its fine, and what I feel is that the cell lose by the method which I use will be less compared to Hamed's suggested method. I just wanted to know, whether its correct or not.

yes, you can do both of the staining in a single protocol

but first stain your surface marker proteins

wash it with pbs

then permeablize the cellular membranes and stain your protein

Also you can use antibody to eliminate non specific binding proteins.

In my opinion, surface staining should be carried out first (prior to fixation) for a number of reasons.. Not least changing the epitopes exposed at the cells surface. Also, fixation can cause unspecific binding of antibodies to surface markers they would normally not recognise without fixation.

Then, you can fix and permeablise your cells to stain for intracellular markers. Fixation will preserve your cell surface staining allowing you to stain within the cell.

So in order, I would carry out the following

Live/Dead stain, using a lysine fixable dye. Not 7AAD, dapi, PI etc..

Surface Stain

Fix/Perm

Intracellular stain

Analyse!