Hello, everybody We have noticed on the list several discussions about surface molecules on adherant cells and the effect of trypsin. Since we had undertaken a few number of experiments we thought that it would be interesting for a larger number of Flowers to have access to some of our results. Our purpose was to check whether trypsin would alter the quantitation of various adhesion molecules on adherant cells. Here the cells we used were Fibroblasts MRC5. When used at appropriate concentration for addherant cells detachment (we use 0.05% trypsin + 0.02% EDTA for up to 15 minutes at 37°C, maximum), it will not modify the expression of these adhesion molecules as shown using the QIFI technology (indirect immunofluorescence staining and quantitative flow cytometry readings). As an example quantitation of integrins CD49b,d,e,f and CD29 on fibroblasts was comparable when detached using either Trypsin -EDTA or Versene (see table 1 below). One may noticed a variation for CD49d although we should certainly repeat our experiment. In our hands cell recovery and viability tends to be poorer using Versene treatment. Table 1: Comparison of quantitation of surface molecules on fibroblasts MRC5 after either Trypsin or versene cell detachment procedures Results expressed as quantitative number of bound MoAbs per cell Monoclonal Antibodies Trypsin Versene Isotypic control IgG2a 1,000 5,100 Isotypic control IgG1 3400 1,000 CD9 545,000 510,400 CD49b 254,500 269,500 CD49d 11,400 22,500 CD49e 197,300 243,000 CD49f 19,250 21,100 CD146 244,150 271,750 CD51 59,900 75,600 CD29 795,495 746,750 Positive control 724,850 778,400 (MoAbs Cocktail reacting on human cells) In another experiment we have quantified integrins on human keratinocytes after trypsin treatment: The results were as follows: Monoclonal antibodies MAb molecules bound per cell CD29 700,000 CD49b 295,000 CD49d 7,500 CD49e 46,500 CD49f 161,000 CD51 41,000 CD9 1,340,000 In addition we noticed a clear difference between the quantitation of CD146 on Fibroblast (> 200,000 sites per cell) and on keratinocytes (< 2,000 sites per cell). In the case someone is looking for more details and specific conditions for endothelial cells we suggest the following paper: Reevaluation of Trypsin-EDTA for Endothelial Cell Detachment before Flow Cytometry Analysis. Mutin, M, George, F, Lesaule, G, Sampol, J. Endothelium, 1996, vol 4, pp 289-295. Michel Canton, PharmD Philippe Poncelet, PhD from BioCytex, Marseille, France
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