Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours

Cytometry
L WebsterG D Wilson

Abstract

Hypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumours. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumour may repopulate faster during treatment. Clinical trials are underway to assess the importance of both these parameters. In this article we describe a method to simultaneously measure hypoxia and proliferation using multiparameter flow cytometry. Hypoxic cells were detected using a bioreductively bound marker with an immuno-recognisable side-chain, NITP and proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation. These parameters were related to cell cycle position by measuring total DNA content with 7-aminoactinomycin D. The data were analysed using single laser excitation on a bench top flow cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypoxic by the criterion of NITP binding. The murine SaF tumour has a relatively constant aneuploid labelling index of 24%. However, the level of aneuploid hypoxia was variable ranging from 0.8 to 40.9% with a mean value of 15.6%. Within the BrdUrd labelled population t...Continue Reading

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Citations

Oct 18, 2000·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·J BussinkA J van der Kogel
May 22, 2003·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Johan BussinkAlbert J van der Kogel
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