Since FITC is a smaller dye it gets into the cell easier than PE. At 300-500g lysing should not be a problem. If your cells are lysing spin at a lower speed for a longer time. It is possible your pipet tips are do not have large enough openings for your cells. Also try pelleting cells in up 10% BSA. The BSA cushions the cells and prevents sticking. On Wed, 19 May 1999, Bharathi Laxman,Ph.D. wrote: > > > Hi, > I want to do an intracellular and extracellular staining of Fas > and I thought of doing it in the following way: > First stain for extracellular Fas, fix the cells, permeabilise and stain > for intracellular Fas using either PE/FITC conjugated antibody > respectively. > Is this a good way to do it? > Also, I wanted to know the protocol to be followed for permeabilisation of > cells as I am using brain tumor cells. Does the protocol differ for cell > type? I have tried to permeabilise using either 70% ethanol, triton x-100 > or saponin but I always tend to lose cells. I would like to know the speed > of pelleting the cells and how one washes after permeabilisation as I lose > cells at this stage. > I appreciate a quick response for this mail > Thank you > Bharathi > > > > > Bharathi L., Ph.D. > Human Medical Genetics Unit > A209 Medical Alumni Building > University of Vermont College of Medicine > Burlington VT 05405. USA > Telephone Lab: 001 802 656 0390 Res: 001 802 658 1518 > FAX: 001 802 656 2641 > E-mail: blaxman@zoo.uvm.edu > >
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