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Simultaneous analysis of mitochondrial activity and DNA content in ehrlich ascites tumor cells by dual parameter flow cytometry

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Summary

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 μg/106 cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.

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Abbreviations

BSA:

bovine serum albumin

CCCP:

carbonyl cyanide m-chlorophenylhydrazone

EAT:

Ehrlich ascites tumor

EGTA:

ethylene glycol bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid

Hepes:

4-(2-hydroxyethyl)-1-piperazineethansulfonic acid

IM:

incubation medium

Rh 123:

rhodamine 123

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Authors and Affiliations

  1. Institut für Physiologische Chemie der Philipps-Universität Marburg, Lahnberge, D-3550, Marburg, Federal Republic of Germany

    T. Hämmerle & M. Löffler

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  1. T. Hämmerle
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  2. M. Löffler
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Dedicated to Professor K.J. Netter on the occasion of his 60th birthday

Enzymes: Ribonuclease (EC 3.1.27.5), Hexokinase (EC 2.7.1.1), Glutamate dehydrogenase (EC 1.4.1.2), Lactate dehydrogenase (EC 1.1.1.28)

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Hämmerle, T., Löffler, M. Simultaneous analysis of mitochondrial activity and DNA content in ehrlich ascites tumor cells by dual parameter flow cytometry. Histochemistry 93, 207–212 (1989). https://doi.org/10.1007/BF00315976

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