> -----Original Message----- > From: Nathalie Tessier [mailto:tessien@ircm.qc.ca] > Sent: Friday, February 08, 2002 8:52 AM > To: Cytometry Mailing List > Subject: [Fwd: Paul Jolicoeur] > > > We are having problems labeling the liver Kupffer cells > (macrophages) > > with different antibodies by FACS. The isotype controls > label the cells > > as intensely as the antibodies . As you know, these cells > are designed > > to actively phagocytose. Is there a trick to get specific labeling > > easily? Hi Nathalie, I would suggest the following steps in your procedure: 1] Try not to use whole Ab to label, better to use F(ab')2 fragments 2] Use an Fc blocking step prior to Ab labeling 3] Keep the cells on ice throughout to prevent Ab internalizaiton 4] always run unlabeled vs. isotype, and then isotype vs test so that the amount of nonspecific labeling is measured for each experiemnt 5] Don't use protease as part of your isolation procedure as this can alter the cell surface good luck, Tom **************************************************************************** * Thomas W. Mc Closkey, Ph. D. Director of Flow Cytometry, North Shore University Hospital Assistant Professor of Pediatrics, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 **************************************************************************** *
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