From: Derek Davies (davies@icrf.icnet.uk)
Date: Thu Dec 28 1995 - 05:13:22 EST
The protocol that I find most useful for permeabilisation for the detection of internal antigens is to use saponin. It is relatively gentle (ie it doesn't put enormous holes in the cytoplasm or the cell membrane) and can be used in conjunction with surface staining or DNA staining. You may have to play about with conditions but a good starting point is to treat cells with 0.3% saponin for about 15mins at room temperature before doing the antigen detection and then to use 0.1% saponin in all subsequent steps. Be careful with the washing steps as it is easy to lose all your cells! We have found that the permeabilisation is best at room temperature and that the saoponin needs to be present all the time as the permeabilisation effect can be reversed. As with all intracellular staining washing is important, but if you are using directly conjugated antibody, this reduces the steps and reduces cell loss. Hope that this is of some use. Derek Derek Davies Imperial Cancer Research Fund London http://www.icnet.uk/axp/facs/davies/index.html On Tue, 26 Dec 1995, Howard Ratech wrote: > > > We are trying to determine if COS cells are expressing a particular > protein after transient transfection. The protein is designed to be > secreted but I am hoping we can find enough of it in the cytoplasm to tell > us the experiment worked before we do a Western blot on the culture > supernatant.We have directly PERCP conjugated mouse monoclonal antibody to > this protein. Do you have any recommended protocol for permeablization, > staining, washing etc.? Since these cells are quite large, is there > anything we should be doing with the FSC and SSC, other than using linear > amplification? We are using a BD FACSCAN with LYSIS software. > > Happy Holidays. > > Howard > >
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