Ron Where did you get the teflon dishes from? I would like to stimulate whole blood and look at cytokine production in the monocytes and lymphocytes, but I am wondering how to retrieve the monos. Virginia Ronald Rabin wrote: > 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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