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> Does anyone have a good protocol for removing (essentially all) surface
> antibody from cells after sorting?
We found (unpublished) that some but not all monoclonal antibodies can be
removed (at least 95%) from viable cells by washing at pH 4.0 for 20 min in
the cold. Keeping the majority of the cells viable at this pH requires
that the suspension be kept strictly at ice temperature, also during the
subsequent pH 7.3 rinse. Some cell functions partially survive this
treatment (CTL-mediated killing, LFA-1 mediated adhesion by JY EBV-LCL),
and others are altered. We added 10 mM citrate to complete medium including
serum, and adjusted the pH to 4.0.
In our hands, incubation at 37 deg in crystalline trypsin was ineffective
at removing antibodies (rat IgG2a monoclonals or mouse IgG1 monoclonals)
from cell surfaces. In fact we found that the presence of a single bound
(and washed) monoclonal Ab protected its antigen from trypsin sufficient to
remove that antigen (in the absence of the antibody). The Ab could be
detected by indirect fluorescence, still bound after a wash in trypsin
inhibitor (also unpublished).
/*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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