As far as bacteria stuck on the outside - I believe you can add crystal violet to your samples to quench any fluorochromes outside of the cells. The problem here is that at least in my experience quite a few bacteria will adhere to the cells in the early stages of phagocytosis and you would lose their contribution to the total numbers. At 10:32 AM 1999-06-01 -0500, Koratich, Mike wrote: > >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 >> > > - Derek Schulze Flow Cytometry and Confocal Microscopy Core Facility Cancer Research Labs Queen's University Kingston, Ontario Canada
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