--- On Mon, 4 Jan 1999 10:59:20 -0500 "Kroeger, Jodi" <KroegerJL@Moffitt.usf.edu> wrote: >Specifically, she is trying to quantitatively assess the apoptotic response >of drug-treated patient PBMC lymphocytes (only) using an Annexin/PI kit. >She cultures the PBMCs for a specified amount of time with IL-2 to activate >them. She then ficolls to remove the dead cells before Annexin staining. >When she brings them to me for flow analysis, we are seeing is a large >population of lymphs, a small population of monos, and a population of what >I believe to be non-lysed RBC's overlapping the apoptotic (various stages) >cells from the PBMCs. This, of course, makes it difficult to distinguish >the apoptotic lymphs from the RBCs and apoptotic monos using light scatter. We have had success using annexin biotin followied by streptavididn APC. This would allow a combination such as CD45 FITC and CD14 PE to distinguish lymphhs. Of course you could also use a subset specific marker such as CD3 depending on what population you were interested in. You could substitute any streptavidin fluor which fits in with the monoclonals you are using. We omit PI for the annexin assay, you should determine if you can do this in your system. If only apoptotic cell death is present, the only info that the PI addsi is early vs. late apoptotic. I would also recommend verifying the results you obtain with the annexin assay by fluorescence microscopy. You can stain cells with PI and look for nuclear changes characteristic of apoptosis. Another good check is to run antother method such as TUNEL on the same sample. What is the reason for the ficooll step to remove dead cells? If the goal is to quantify the percentage of the population which responded by undergoing apoptosis this is skewing the results. For a comparison of methods applicable to PBMC I refer you to AIDS Res Hum Retro, 14: 1413-1422, 1998. Good luck, Tom -------------------------------------------------------- Thomas W. Mc Closkey, Ph. D. Director, Flow Cytometry North Shore University Hospital 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 1/6/99 5:28:32 PM E-mail: thomasm@nshs.edu --------------------------------------------------------
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:52:53 EST