permeabilization without killing cells

Howard Shapiro hms at shapirolab.com
Tue Oct 30 15:30:46 EDT 2007


Joern Schmitz wrote:

> I am not sure how one would get specific intracellular staining by
> electroporation. Also, I don't know which dyes would need to be coupled
> to the antibodies of interest to perform specific intracellular staining
> by electroporation and detection by flow cytometry.
> 
Hi Joern-

Antibody (FITC-labeled) was added to the electroporation buffer, and
cells were washed while still permeabilized. The staining looks pretty
specific in the paper.

> I guess the capturing of secreted molecules on the cell surface would be
> a much better technique. There are several papers from Rudi Manz. Here
> is one:
> 
> 
> */Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1921-5. /*
> 
> */Analysis and sorting of live cells according to secreted molecules,
> relocated to a cell-surface affinity matrix.Manz R, Assenmacher M,
> Pflüger E, Miltenyi S, Radbruch A./*
> 
> Also, Miltenyi Biotec sells capture kits.

I didn't mention the capture matrix technique (which I mentioned
favorably in the 4th Edition of P.F.C.) because the original posting
inquired about an antibody to an intracellular, rather than a secreted
protein. The capture matrix is obviously a better idea for detecting
secreted proteins because it is much less likely than electroporation to
perturb the cell.

-Howard





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