Koratich, In reply to your question on phagocytosis by flow, you could probably modify the following protocol which I learnt in a flow cytometry course at Sheffield: 1. Use siliconised glass tubes 2. Add 40 ul of a 2.5% suspension of fluorescent 1 um diameter microspheres (Polysciences) to the macrophages suspended in 1.9 mls PBS. Incubate at 37 C for 60 min. 3. Gently pipette the suspension onto 1 ml fetal calf serum and centrifuge to generate a cell pellet (200g) 4. Aspirate the supernatant containing free beads and discard 5. Resuspend the cells in 1-2 mls PBS+0.1% trypsin (type IIIs, Sigma) and 5nMol EDTA (ie equal parts stock tissue culture Trypsin/Versine). Incubate at 37C for 10 min to detach adherent but non phagocytosed beads 6. Repeat step 3. 7. Wash cells X2 in cold PBS 8. Analyse by flow John ------------------------------------------------------------------------ Dr. John N. Waitumbi c/o Dr Jose A. Stoute, USAMRU, Unit 64109, Box 401 APO AE 09831-4109 Kenya Phone +254 35 22942 Email:waitumbi@net2000ke.com Fax +254 35 22903 > 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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