Dee, There are a few answers to your question, depending on what you are looking for. If you are purifying monocytes from whole blood, there are pretty good separation reagents available, for example from Stem cell technologies. There are protocols using Percoll that give you good purity as well. Then you can use them for experiments, and let them become macrophages with time, or accelerate with M-CSF. Elutriation is great if you have it available to you. All these will yeild more cells than adherence. If there is some reason that isolating by adherence is important, the trick I used long ago was to plate the PBMC on large petri dishes that are not tissue culture treated. The monocytes stuck and spread well, but could be washed off with cold PBS in the absence of calcium (I would throw in some EDTA). If you let them sit for too long, they will stick too strongly though; they appear to lay down their own matrix. I tried pure teflon dishes and couldn't get them off after a few days. ron on 2/21/02 9:09 PM, Dee Harrison-Findik at duygu.harrison-findik@imvs.sa.gov.au wrote: > > Dear Flowers > I am isolating macrophages from human blood by the simple adherence > technique. > I am having difficulties to harvest the adhered macrophages by conventional > trypsinisation. Majority are still stuck to the TC. flask. Cell scraping > does not yield very happy cells, either. > Is there any specific reagent or technique which will lift off all (or > majority) of the adhered macrophages as viable as possible ? > Any input is greatly appreciated. > Cheers > Dee > > > Ronald L. Rabin, M.D. Senior Staff Fellow Laboratory of Immunobiochemistry DBPAP/OVRR Center for Biologics Evaluation and Research U.S. Food and Drug Administration 29 Lincoln Drive (MSC-4555) Building 29, Room 129 Bethesda, MD 20892-4555 phone: 301.496.8806 fax: 301.402.5177 email: rr84g@nih.gov
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