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. 1981 Apr;78(4):2383–2387. doi: 10.1073/pnas.78.4.2383

Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation.

Z Darzynkiewicz, L Staiano-Coico, M R Melamed
PMCID: PMC319350  PMID: 6941298

Abstract

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day of cell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake of rhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.

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Selected References

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

  1. Bauer K. D., Dethlefsen L. A. Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy. J Histochem Cytochem. 1980 Jun;28(6):493–498. doi: 10.1177/28.6.6156196. [DOI] [PubMed] [Google Scholar]
  2. Bradley D. F., Wolf M. K. AGGREGATION OF DYES BOUND TO POLYANIONS. Proc Natl Acad Sci U S A. 1959 Jul;45(7):944–952. doi: 10.1073/pnas.45.7.944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coulson P. B., Bishop A. O., Lenarduzzi R. Quantitation of cellular deoxyribonucleic acid by flow microfluorometry. J Histochem Cytochem. 1977 Oct;25(10):1147–1153. doi: 10.1177/25.10.72097. [DOI] [PubMed] [Google Scholar]
  4. Darzynkiewicz Z., Evenson D. P., Staiano-Coico L., Sharpless T. K., Melamed M. L. Correlation between cell cycle duration and RNA content. J Cell Physiol. 1979 Sep;100(3):425–438. doi: 10.1002/jcp.1041000306. [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., 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]
  7. 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]
  8. Darzynkiewicz Z., Traganos F., Sharpless T., Melamed M. R. Lymphocyte stimulation: a rapid multiparameter analysis. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2881–2884. doi: 10.1073/pnas.73.8.2881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. INMAN D. R., COOPER E. H. ELECTRON MICROSCOPY OF HUMAN LYMPHOCYTES STIMULATED BY PHYTOHAEMAGGLUTININ. J Cell Biol. 1963 Nov;19:441–445. doi: 10.1083/jcb.19.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson L. V., Walsh M. L., Chen L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980 Feb;77(2):990–994. doi: 10.1073/pnas.77.2.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. LERMAN L. S. The structure of the DNA-acridine complex. Proc Natl Acad Sci U S A. 1963 Jan 15;49:94–102. doi: 10.1073/pnas.49.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sharpless T., Traganos F., Darzynkiewicz Z., Melamed M. R. Flow cytofluorimetry: discrimination between single cells and cell aggregates by direct size measurements. Acta Cytol. 1975 Nov-Dec;19(6):577–581. [PubMed] [Google Scholar]
  13. Traganos F., Darzynkiewicz Z., Sharpless T., Melamed M. R. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J Histochem Cytochem. 1977 Jan;25(1):46–56. doi: 10.1177/25.1.64567. [DOI] [PubMed] [Google Scholar]

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