We never scrape adherent cells off the flask for flow since so many are shredded and cannot be gated by FSC or SSC. Additionally, since CD14 is shed when cells are cultured, I feel the likelihood of trypsin cleaving it is high. Of course details would depend on the anti-CD14 antibody clone and where that antibody mapped. To detach any adherent cell line from a flask when we want to stain for flow, we use 5mM EDTA in PBS. After removing the media, cover the monolayer with just enough EDTA/PBS and then start watching the cells round up in the microscope. This can take 30 seconds (HeLa and 293T cells) or 15 minutes (day 15 macrophages). Remember that almost all cells hate EDTA, so after they lift up, immediately pipette them out of the dish/flask (and gently scrape if you need to AFTER they round up), then quickly resuspend them in media and centrifuge them out of the EDTA. About trypsin and other receptors: CD4 is always cleaved, but above the epitope for OKT4. Therefore, you can use OKT4 on trypsinized cells and still have CD4 reactivity (even though D1-D2 is gone). About chemokine receptors, I have found their epitopes amazingly resistant to trypsin. Particularly, CXCR4 retains significant amounts of staining by 12G5 as well as several antibodies of clones from R&D System. This is probably due to the epitopes being "buried" in the internal loops where trypsin does not cleave. Good luck. JDT ********************* Julie Davis Turner Postdoctoral fellow Centers for Disease Control & Prevention NCID/DASTLR/Tb Atlanta, Georgia USA
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:37 EST