Cytometry 12:68-76 (1991)
0 1991 Wiley-Liss, Inc.
Simultaneous Quantification of cmyc Oncoprotein, Total Cellular Protein, and DNA Content Using Multiparameter Flow Cytometry' Herbert H. Engelhard 111, Jeri L. Krupka, and Kenneth D. Bauer Department of Pathology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611. Received for publication May 10, 1990; accepted July 24, 1990
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 technique 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, SR101, and DAPI. FITC-labelled antibody coupled with indirect immunofluorescence was used to quantify the c-myc oncoprotein, whereas SRlOl and DAPI were used to measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of cmyc oncoprotein were compared to densitometric readings of p64c-myc.SRlOl protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements correlated well
Total cellular protein varies in relation to cell growth and proliferation. Early studies comparing noncycling and stimulated lymphocytes indicated a 1020-fold increase in protein synthesis and a three-fold increase in dry cellular mass (7711719).Crissman et al. have additionally shown a n approximately two-fold difference in protein between GI- and G,-phase using exponentially growing Chinese hamster ovary cells and dual-parameter flow cytometry (FCM) to simultaneously analyze total cellular protein and DNA content (10). Recently, considerable interest has focused upon the use of monoclonal antibodies and immunocytochemical methods to examine modulations of specific proteins in relation to cell cycle phase. Such analyses are commonly pursued in conjunction with cell separation methods to enrich for cell cycle subpopulations followed
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 Fg/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 1991 Wiley-Liss, Inc.
Simultaneous Quantification of cmyc Oncoprotein, Total Cellular Protein, and DNA Content Using Multiparameter Flow Cytometry' Herbert H. Engelhard 111, Jeri L. Krupka, and Kenneth D. Bauer Department of Pathology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611. Received for publication May 10, 1990; accepted July 24, 1990
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 technique 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, SR101, and DAPI. FITC-labelled antibody coupled with indirect immunofluorescence was used to quantify the c-myc oncoprotein, whereas SRlOl and DAPI were used to measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of cmyc oncoprotein were compared to densitometric readings of p64c-myc.SRlOl protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements correlated well
Total cellular protein varies in relation to cell growth and proliferation. Early studies comparing noncycling and stimulated lymphocytes indicated a 1020-fold increase in protein synthesis and a three-fold increase in dry cellular mass (7711719).Crissman et al. have additionally shown a n approximately two-fold difference in protein between GI- and G,-phase using exponentially growing Chinese hamster ovary cells and dual-parameter flow cytometry (FCM) to simultaneously analyze total cellular protein and DNA content (10). Recently, considerable interest has focused upon the use of monoclonal antibodies and immunocytochemical methods to examine modulations of specific proteins in relation to cell cycle phase. Such analyses are commonly pursued in conjunction with cell separation methods to enrich for cell cycle subpopulations followed
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 Fg/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
