Simultaneous quantification of c-myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry.
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 SR101 and DAPI were used to measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of c-myc oncoprotein were compared to densitometric readings of p64c-myc. SR101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements correlated 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 micrograms/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 less than 0.05), a relative increase in the proportion of G0/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 or cytostatic drugs.
Engelhard
H H
HH
Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.
Krupka
J L
JL
Bauer
K D
KD
D002453:Cell Cycle; D002460:Cell Line; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007211:Indoles; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis* / Q000378:metabolism; D012235:Rhodamines; D013861:Thiocyanates; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism / Q000473:pathology
D005452:Fluoresceins; D007211:Indoles; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D012235:Rhodamines; D013861:Thiocyanates; C007293:DAPI; D004247:DNA; D003513:Cycloheximide; C050829:sulforhodamine 101; D016650:Fluorescein-5-isothiocyanate