Hallo Mike, I worked on phagocytosis of macrophages, too. I used labelled 1µm FITC beads etc. or unlabelled beads up to 10µm. The way to obtain cells with high viability (>95%) was to use culture plates without!!! TC for adhearence (works fine) and to remove them with ice cold 1% EDTA/PBS (incubation: 10' at 4°C). Good luck for your work Joerg ----------------------------------- Joerg Hildmann Institut fuer Immunologie Universitaet Mainz - Germany ----------------------------------- You wrote: > >Hi all, > >Does anyone have a good protocol for doing phagocytic activity of peritoneal >macrophages using FITC beads by flow. We are currently harvesting the >macrophages by lavage and adherence to 6-well TC plates for 2 hours in DMEM >w/o serum. We then wash and add DMEM w/ 10% FBS at let them incubate ON. >We then wash and add 1E8 beads, which is roughly a 100:1 bead to cell >ratio. This is incubated for 90 mins. and then washed to remove most of the >free beads. This all works fairly well and the cells look good under the >scope. Our problem is how to get the cells to detach so we can run them on >the flow without causing disruption of the membrane and loss of beads. We >are currently using 0.4% EDTA with poor results. We have found a protocol >that uses dispase and will try that next. Any ideas on other things to try >would be appreciated. Further, any suggestions on the method in general are >also welcome. > >TIA, > >Mike Koratich >Cell Biology and Immunology Group >Serquest >Southern Research Institute >Birmingham AL 35205 > >
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