intracellular FACS staining - HELP!

tronni at my-deja.com tronni at my-deja.com
Wed Feb 23 03:28:42 EST 2000


In article <20000216035046.09862.00000089 at ng-fy1.aol.com>,
  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
follow
> this with a first-step (biotinylated polyclonal rabbit antisera to the
antigen,
> 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
lessen
> this intensity, I added a step before my original first step: right
after
> permeabilizing the cells I would block for 30 min with 2% rabbit
serum.  This
> did not help at all.

Hi,

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




Sent via Deja.com http://www.deja.com/
Before you buy.




More information about the Methods mailing list