Intracellular proteins on live cells - (Jun/25/2008 )

Hello - I am trying to do flow cytometry on live cells, but I am looking at intracellular proteins. I previously tried to do intracellular staining by this protocol:

1. trypsin cells, count
2. permeabilize with Triton-X
3. primary, then secondary

Problems I faced were loss of cells (as I was staining in a suspension) and cells clumping together after a while, and the staining never really did work. Do you have alternative suggestions I can try?

When people do sorting to obtain a more pure population of cells, they have to do it on live cells, right?

Thanks,
- Eli

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Hi Eli,

Yes, to sort for a more 'pure' population of cells they have to be alive so you can culture them again after you have sorted.

If your protein of interest is cytosolic, why don't you try with a less "harsh" detergent such as digitonin?

This paper by Waterhouse et al Methods Mol Biol. 2004;284:307-13. does something similar looking at cytochrome c release from mitochondria.

Hope this helps

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The 2 main companies that make reagents for flow cytometry (BD and eBioscience) make buffer kits for intracellular staining. i think that they use either saponin or maybe methanol...probably saponin. either way, dig might be less harsh than Triton (sledgehammer!!), but the kits are already troubleshot and should be easy to use the first time. If you wish to culture cells that you have sorted following intracellular staining....it won't happen because most protocols have some sort of fixation which would lock the cells as they were at the time of sorting.

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I use 0.5% saponin, and it works fine.
Fix your cells with PFA 4%. Incubate your cells with antibodies with 0.5% saponin, 1 hour 4°C
wash twice with saponin, once without saponin.

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