Open AccessSimultaneous quantification of c‐ myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry
Author(s) -
Engelhard Herbert H.,
Krupka Jeri L.,
Bauer Kenneth D.
Publication year - 1991
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990120110
Subject(s) - cycloheximide , dapi , flow cytometry , biology , microbiology and biotechnology , cell cycle , dna , immunofluorescence , cell , protein biosynthesis , apoptosis , antibody , biochemistry , immunology
Variations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a techniaque is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual‐laser (uv and 488 nm) excitation and three fluorescent dyes: FITC, SR 101, and DAPI. FITC‐labelled antibody coupled with indirect immunofluorescence was used ot quantify the c‐ myc oncoprotein, whereas SR 101 and DAPI were used ot measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of c‐ myc oncoprotein were compared to densitometric readings of p64 c‐ myc SR 101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements corrlated well with those obtained by the biochemical methods. The usefulness of the technique was further examined following treatment of exponentially growing HL‐60 cells with 2.5 μg/ml cycloheximide for 0 to 12 h. Cycloheximide treatment was found to cause a significant decrease in c‐ myc oncoprotein content within 2 h (P<0.05), a relative increase in the proportion of G 0 /G 1 cells and a modest decrease in total cellular protein. This technique appears to provide a rapid, quantitative approach, useful for investigating alterations in cellular growth balance occurring with cell differentiation, neoplastic transformation, or cell treatment with radiation of cytostatic drugs.