Our protocol for intracellular staining uses 4% paraformaldehyde + 1% triton x-100 to fix the cells, and incubate with primary ab for 1 hr. We have tried this with several cancer cell lines and the flow cytometry results seem ok.

What about if I want to grow the cell after the sorting ? is there any proper protocol to follow?

Hi Abdulghani

First thing - you cannot do intracellular staining on live cells. You need to permeabilize your cell membranes to do intracellular stain.

If you want to sort your cells and grow them, I would recommend you to do a literature search to find a suitable surface marker that is expressed in your intracellular positive cells. This could be an alternative strategy many people use.

Example. I work with muscle satellite cells (SC). SC's are identified by the expression of transcription factor PAX7 ( intracellular protein), which is not possible in live cells, so I looked for unique surface markers that are expressed in PAX7 +ve cells and not expressed in PAX7 -Ve cells. BY this I was able to sort the live cells with the surface marker and ended up getting PAX7 +ve cells. ( this can be standardized using immunofluorescence or immunocytochemistry )

Before doing FACS I would suggest you to check the antibody reactivity by doing a Immunocytochemistry.

Thank you so much for your good knowledge Ashis.I will consider what you recommend.