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
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:30 EST