intracellular FACS staining - HELP!

tronni at tronni at
Wed Feb 23 03:28:42 EST 2000

In article <20000216035046.09862.00000089 at>,
  tiey at aol.comjunkbloc (Kiley R. Prilliman) wrote:
> Hi,
> I am trying to screen some transfectants expressing an intracellular
protein by
> FACS but have been having some problems.  I am permeabilizing the
cells (mouse
> endothelial cell line) with 0.25% saponin.  In the beginning, I would
> this with a first-step (biotinylated polyclonal rabbit antisera to the
> 1:500, 30 min), wash, and then a second step (streptavidin-PE, 1:500,
20 min).
> However, my controls stained as brightly as anything else!  To try to
> this intensity, I added a step before my original first step: right
> permeabilizing the cells I would block for 30 min with 2% rabbit
serum.  This
> did not help at all.


I have been doing quite a lot of intracellular staining of cytokines
in human and mouse macrophages. There are some tricks you may want
to try. I don't know if you fixed cells first with e.g. 4% PFA.
I never did permeabilization without fixing cells first. My
permeabilization buffer contains 1% FCS and 0.1% (w/v) saponin in PBS
plus 0.1% sodium azide.

I always stain in PBS containing 3% FCS and 0.1% sodium azide.

As a negative control I would stain untransfected and transfected
cells with an irrelevant primary rabbit Ab.

If your cells express Fc receptors that can bind antibodies
in an unspecific manner you may try staining on ice or preblocking Fc
receptors with excess of irrelevant rabbit Ab.

Of course your primary Ab may be cross-reacting with something
in your endothelial cells; you may want to try a different Ab.

Pharmingen's catalog has a nice technical section of intracellular FACS
I have found useful.

Good luck!

Tapani Ronni, Ph.D.
Postdoctoral fellow, UCLA/HHMI

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