Hi everybody, I was planning to not answer this question, but Howard sent me a note asking me to address the labeling of cytoplasmic antigens. We were successful in labeling cytoplasmic p21ras using electroporation a few years ago. We removed the adherent cells with three washes of Tyrode's Ca and Mg free medium and a very brief exposure to trypsin, which incidentally, might solve Pierre's first problem, labeling surface integrins. We placed the cells in PBS along with the labelled antibody of interest, immediately zapped them, can't remember offhand the voltage, something like 250, i think, then immediately placed them back in serum containing medium. The pores in the membranes closed fast, making the cells impermeable to even PI. We went on to sort, culture, and do metastasis assays with the various populations with verious levels of p21. This work was published in Cytometry in 1991 and in J Imm Meth later. Oh and a comment on art vs science for Mario----try doing confocal, the art end of the equation is huge! And such fun!! And hey - no dot plots. Deb Berglund Montana State University On Thu, 2 Oct 1997, P. VAIGOT wrote: > > Dear group > > Actually we try to stain dissociated epithelial cells and we have two kinds > of problem. > - First, staining of cells in suspension with antibody anti integrin > subunit seems very difiicult to detect and is not reproductible. > > - Second, does anybody have some practice of VITAL staining of cells with > an intracellular cytoplasmic marker such as cytokeratin? > > Does anybody have some experiment which would help us? > > > Thanks > > PIERRE > > >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:50:12 EST