The use of dansyl lysine to assess heat damage and thermotolerance of normal tissues.

R Sekiguchi, G C Rice, G M Hahn

Index: Int. J. Radiat. Oncol. Biol. Phys. 14(5) , 983-8, (1988)

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Abstract

The fraction of cells excluding the fluorescent dye dansyl lysine has previously been shown to correlate well with heat-induced cell killing in a variety of mammalian cell lines and in murine tumors in vivo. Here we evaluate the usefulness of dansyl lysine as a probe for assessment of thermal damage and for measuring the kinetics of thermotolerance development and decay in murine normal tissues. Skin cells were heated in vivo with an initial treatment of 44 degrees C for 20 min by local radiofrequency. Bone marrow cells were heated at 42.5 degrees C for 20 min by whole body water bath immersion. Cell suspensions were prepared, heated in vitro for various lengths of time at 44 degrees C (skin) or 43 degrees C (bone marrow), and scored for the fraction of dansyl lysine-excluding cells. Skin and bone marrow cells expressed maximum thermotolerance by 8 and 6 hr, respectively and returned to normal heat sensitivity by 48 and 146 hr, respectively. The assay was not useful with skeletal muscle and liver, as we were not successful in obtaining viable, dansyl lysine-excluding cells from these tissues. Also, in our hands red blood cells, normal human leukocytes, mouse spleen and thymus cells all failed to stain dansyl lysine even after extreme heating. Dansyl lysine staining, particularly when combined with flow cytometry analysis, has been shown to be a useful method for assessing thermal damage and thermotolerance relatively rapidly in all tumor systems tested to date, and, as shown here, may possess utility in measuring similar endpoints for certain nonclonogenic normal tissues.