ISSN:
0173-0835
Keywords:
Two-dimensional polyacrylamide gel electrophoresis
;
Antibodies monoclonal diagnostic use
;
Colonic neoplasms
;
Rectal neoplasms
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti-cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two-dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 × 107 viable normal and tumoral cells before fixation, and up to 4 × 106 cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin-positive cells in the samples was the following (mean, Cl 5-95%): mucosa 29.5% (8.9-66.7%), tumor 44.3% (6.6-94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150180346
Permalink
