Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA.
We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.
Rosette
C D
CD
Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California 94550.
DeTeresa
P S
PS
Pallavicini
M G
MG
D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis*; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D004261:DNA Replication / Q000502:physiology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006358:Hot Temperature; D011419:Propidium; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins / Q000032:analysis; D013861:Thiocyanates
D005452:Fluoresceins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D013861:Thiocyanates; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; D016650:Fluorescein-5-isothiocyanate