Simultaneous quantification of c‐myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry

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Abstract
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 p64c‐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 (P0/G1 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.