Hello, I know that staining specificity and isotype controls are currently discussed on this mailing list but I still have a question... A phD student is working on adherent cell line (SKNMC, human neuroblastoma cell line (HTB10)). he detached them using EDTA. He wants to study a protein expression following shRNA treatment. I attached the results because we need help for results interpretation... Here is the problem we encounter : He is doing a 2 steps staining. - he has no aspecific staining due to his secondary antibody. - he can see 2 peaks with his isotype control : I can't explain it... What does this mean?? - the fluorescence of his antibody-specific staining is above the one obtained with his isotype control but can we consider that it is specific and compare the results obtained in the different conditions? When you look at the histograms, is it possible to conclude that we really have less protein following the shRNA treatment compared to the control-shRNA? I am puzzled... (You can also give me your advice on the FS/SS dotplot, I think it would be better to increase his FS settings) Thank you very much for your help. Julie Bertout Flow cytometry core Institut Pasteur de Lille France This attachment - 'specific staining.pdf' - 570.25 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/b03bd15e65accb293fe142dff5072c9f931a2f6a.pdfReceived on Fri Apr 18 14:18:00 2008
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