This is a Thank You to all the kind Flowers that responded to my original question found below. What I have now adopted is using Opsonized FITC-E.coli which I mix with the cells in a tube and rock for 1 hour at 37C. The cells are washed and analyzed immediately. By using a cell control and a bacteria control it is possible to exclude the clumped bacteria from analysis. I have tried using an anti-FITC to quench the bacteria that may be stuck to the outside of the cell but have had very little luck. When examining the cells under the scope I find very few free bacteria and those that are present are clumped and easily removed from analysis. I have also seen very few bacteria stuck to the outside of cells. Further, the cells that have bacteria stuck to them also have bacteria inside. While I still have some work to do on this assay, I have a very good jump start now. Thanks Again, > Mike Koratich > Cell Biology and Immunology Group > Southern Research Institute > Birmingham AL 35205 > > ---------- > From: Koratich, Mike > Sent: Thursday, May 20, 1999 1:24 PM > To: Cytometry Mailing List > Subject: Phagocytosis by Flow > > > 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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