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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=B0C=
,
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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web