Flow Cytometry Intracellular and dead cell staining - (Dec/13/2006 )

Has anyone got a method or an idea as to how I would stain for dead cells after I have permeablised cells for intracellular antigen staining? With cell surface antigens I use PI to gate out dead cells but am unable to use this with permablised cells. Also, I start by fixing with a PFA based fixative - so surely all the cells will be dead anyway?!?

I've thought of doing a cell viability assay (count) with trypan blue before I begin the staining for flow cytometry to give a percentage of live/dead cells - does anyone know if this is good enough?

Any advice would be great!

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I have a similar question - Can I stain for apoptotic cells (Annexin?) plus an antibody for an intracellular protein?

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I know that you can combine a cell surface stain with Annexin, but I'm unsure as to whether you can do intracellular staining. With annexin you're looking at externalisation of PS residues on the membrane, so during the process of permeablising the membrane to get the intracellular stain in you may affect these residues. You could always try and compare the annexin data to cells that haven't had the additional stain and if they are the same then you know it works. It would be intersting to know if it does!

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