A novel flow cytometric method for the quantification of p53 gene expression.
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.
Filippini
G
G
Bone Marrow Transplantation Laboratory, La Carità Hospital, Locarno, Switzerland.
Griffin
S
S
Uhr
M
M
Eppenberger
H
H
Bonilla
J
J
Cavalli
F
F
Soldati
G
G
D000230:Adenocarcinoma; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D007938:Leukemia; D008223:Lymphoma; D009154:Mutation; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D016159:Tumor Suppressor Protein p53