Dear Dr Sukwan Handali, As a possible reply to your question #3, I would suggest you have a look to the 2 following papers from Mutin M. et al. 1/ Reevaluation of Trypsin-EDTA for endothelial cell detachment before flow cytometry analysis (1996), Endothelium, 4:289-295. 2/ Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules (1997), Tissue Antigens, 50:449-458. Because they used a precise immuno-cytometric method for quantitation of cell-surface antigens/receptors on endothelial cells, they wanted to check that their optimized method of detaching the adherent cells was not modifying the level of expression of adhesion molecules (including ICAM1). Their receipie is the following : 0.05% trypsin-0.02% EDTA for 30 SECONDS at 37°C. Their study shows the kinetics of decrease and demonstrates that trypsin can be used, even in the most demanding applications (absolute quantitation), provided the time allowed for trypsin incubation is kept under 2 minutes. When working on skin cells (primary explants of fibroblats and keratinocytes) for the quantitation of integrins (CD49a,b,e and CD29), we have observed that harvesting the adherent cells before confluency (about 80% confluency) was a perequisite to recover the cells in a good yield with no loss of expression as compared to EDTA alone (also called "Versène" in french). I hope these small but successful tricks may help you succeed although we never tried them on endothelial cells from monkeys. Regarding question 4, I checked our files to see that we did test our anti-ICAM-1 MAb (clone F4-31C2, IgG1) on monkey leukocytes. It turned out to work well on baboon leukocytes but was negative on the 2 tested Rhesus monkeys (Macacca Mulata). I also remember from the Leukocyte Typing workshops that researchers at TNO (Rotterdam, NL) have an extended experience in phenotyping monkeys. Margaret Jonker was part of the team , you may have a Medline search on her name. Hope this helps. Philippe Poncelet, PhD BioCytex, Marseilles (F) "Sukwan Handali" <shandau@mailhost.tcs.tulane.edu> le 12/06/2000 16:50:26 Pour : Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> cc : (ccc : MailingList/general/Biocytex) Objet : Mabs against ICAM-1/CD54 in rhesus monkey To whom it may concern! I am a student at Dept. Tropical Medicine, Tulane University. I am detecting ICAM-1/CD54 from rhesus monkey brain endothelial cells. As my positive control, human brain endothelial cells. I have detected ICAM-1 in human brain endothelial cells (constitutive and upregulated by TNF), but I failed to detect ICAM-1 in rhesus monkeys. The MAbs that I have used: 84H10 (Immunotech) and LB-2 clone (BD). LB-2 clone according to BD will cross-react to ICAM-1 of rhesus monkey (based on a web-site at Univ. Florida). So, my questions: 1. what should I do so that I could detect ICAM-1 from rhesus monkey? 2. is trypsinization will destroy ICAM1 from rhesus monkeys? (my positive control is trypsinized and works well) 3. I have tried to detach my cells with EDTA 1 mM in PBS and it did not work. Will Sigma Cell Detachment Solution will work? Or, if somebody knows how to detach this kind of sticky cells (without scrapping and trypsin) and still preserve ICAM-1? 4. is there somebody who has work with ICAM-1 in rhesus monkey? I wonder if the researcher(s) who put the information on U Florida website could share the procedure! Thanks. Sukwan Handali
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