Open AccessA novel flow cytometric method for the quantification of p53 gene expression
Author(s) -
Filippini Giuditta,
Griffin Sayuri,
Uhr Mario,
Eppenberger Hans,
Bonilla Josè,
Cavalli Franco,
Soldati Gianni
Publication year - 1998
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19980301)31:3<180::aid-cyto5>3.0.co;2-p
Subject(s) - flow cytometry , microbiology and biotechnology , cell culture , monoclonal antibody , biology , immunocytochemistry , antibody , cell , mutant , immunofluorescence , cytometry , gene , biochemistry , immunology , genetics , endocrinology
We describe a rapid and simple flow cytometry technique for the detection and quantification of p53 in several human cell lines, including an adenocarcinoma cell line (SW 626) having a mutant (m) p53, and a pre‐B leukemia cell line (NALM‐6) having wild‐type (wt) p53. By introducing a second antibody coupled to RPE‐fluorescence, the discrimination between control and specific peaks was improved over that achieved with methods used previously. To quantify the content of p53 molecules in the cells, we used a series of beads with the capability to bind mouse monoclonal IgG antibodies. p53 cell content, expressed as antibody binding capacity (ABC), was directly quantified from logarithmic scattergrams; the results were reproducible in all cell lines tested. Flow cytometric results were compared with those of a standard immunocytochemistry method routinely used for the detection of p53 in cells, and found to be in correlation. Furthermore, cytometric data also reflect ELISA determinations. In summary, we showed for the first time that (i) p53 can be clearly detected by flow cytometry in various cell lines, (ii) p53 can be quantitated in terms of number of molecules per cell, and (iii) it can be easily monitored as a function of time after stimulation. Cytometry 31:180–186, 1998. © 1998 Wiley‐Liss, Inc.