Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Jul 1;101(1):141–147. doi: 10.1083/jcb.101.1.141

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry

PMCID: PMC2113613  PMID: 2409095

Abstract

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three- color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein-fluorescein isothiocyanate (green), and RNA-bound pyronin Y (red) fluorescence measurements were correlated as each stained cell intersected three spatially separated laser beams. The analytical scheme provided sensitive and accurate fluorescence determinations by minimizing the effects of overlap in the spectral characteristics of the three dyes. Computer analysis was used to generate two-parameter contour density profiles as well as to obtain numerical data for subpopulations delineated on the basis of cellular DNA content. Such determinations allowed for analysis of RNA to DNA and RNA to protein ratios for cells within particular regions of the cell cycle. The technique was used to study the interrelationship of DNA, RNA, and protein contents in exponentially growing Chinese hamster ovary cells as well as in cell populations progressing the cell cycle after release from arrest in G1 phase. The sensitivity of the method for early detection of conditions of unbalanced growth is demonstrated in the comparison of the differential effects of the cycle-perturbing agent, adriamycin, on cells treated either during exponential growth or while reversibly arrested in G1 phase.

Full Text

The Full Text of this article is available as a PDF (670.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arndt-Jovin D. J., Jovin T. M. Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem. 1977 Jul;25(7):585–589. doi: 10.1177/25.7.70450. [DOI] [PubMed] [Google Scholar]
  2. Crissman H. A., Steinkamp J. A. Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol. 1973 Dec;59(3):766–771. doi: 10.1083/jcb.59.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Crissman H. A., Van Egmond J., Holdrinet R. S., Pennings A., Haanen C. Simplified method for DNA and protein staining of human hematopoietic cell samples. Cytometry. 1981 Sep;2(2):59–62. doi: 10.1002/cyto.990020204. [DOI] [PubMed] [Google Scholar]
  4. Darzynkiewicz Z., Crissman H., Traganos F., Steinkamp J. Cell heterogeneity during the cell cycle. J Cell Physiol. 1982 Dec;113(3):465–474. doi: 10.1002/jcp.1041130316. [DOI] [PubMed] [Google Scholar]
  5. Darzynkiewicz Z., Evenson D., Staiano-Coico L., Sharpless T., Melamed M. R. Relationship between RNA content and progression of lymphocytes through S phase of cell cycle. Proc Natl Acad Sci U S A. 1979 Jan;76(1):358–362. doi: 10.1073/pnas.76.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Darzynkiewicz Z., Sharpless T., Staiano-Coico L., Melamed M. R. Subcompartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6696–6699. doi: 10.1073/pnas.77.11.6696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Darzynkiewicz Z., Traganos F., Melamed M. R. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry. 1980 Sep;1(2):98–108. doi: 10.1002/cyto.990010203. [DOI] [PubMed] [Google Scholar]
  8. Darzynkiewicz Z., Traganos F., Sharpless T., Melamed M. R. Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res. 1975 Oct 1;95(1):143–153. doi: 10.1016/0014-4827(75)90619-9. [DOI] [PubMed] [Google Scholar]
  9. Pardee A. B., Dubrow R., Hamlin J. L., Kletzien R. F. Animal cell cycle. Annu Rev Biochem. 1978;47:715–750. doi: 10.1146/annurev.bi.47.070178.003435. [DOI] [PubMed] [Google Scholar]
  10. Pollack A., Prudhomme D. L., Greenstein D. B., Irvin G. L., 3rd, Claflin A. J., Block N. L. Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green. Cytometry. 1982 Jul;3(1):28–35. doi: 10.1002/cyto.990030108. [DOI] [PubMed] [Google Scholar]
  11. Salzman G. C., Wilkins S. F., Whitfill J. A. Modular computer programs for flow cytometry and sorting: the LACEL system. Cytometry. 1981 Mar;1(5):325–336. doi: 10.1002/cyto.990010505. [DOI] [PubMed] [Google Scholar]
  12. Shapiro H. M. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry. 1981 Nov;2(3):143–150. doi: 10.1002/cyto.990020302. [DOI] [PubMed] [Google Scholar]
  13. Steinkamp J. A., Stewart C. C., Crissman H. A. Three-color fluorescence measurements on single cells excited at three laser wavelengths. Cytometry. 1982 Jan;2(4):226–231. doi: 10.1002/cyto.990020405. [DOI] [PubMed] [Google Scholar]
  14. Tanke H. J., Nieuwenhuis I. A., Koper G. J., Slats J. C., Ploem J. S. Flow cytometry of human reticulocytes based on RNA fluorescence. Cytometry. 1981 Mar;1(5):313–320. doi: 10.1002/cyto.990010503. [DOI] [PubMed] [Google Scholar]
  15. Tobey R. A., Crissman H. A., Oka M. S. Arrested and cycling CHO cells as a kinetic model: studies with adriamycin. Cancer Treat Rep. 1976 Dec;60(12):1829–1837. [PubMed] [Google Scholar]
  16. Tobey R. A., Ley K. D. Isoleucine-mediated regulation of genome repliction in various mammalian cell lines. Cancer Res. 1971 Jan;31(1):46–51. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES