Use of N-σ-dansyl-L-lysine and flow cytometry to identify heat-killed mammalian cells

Abstract

We have employed the nontoxic fluorescent membrane probe, N-σ-dansyl-L-lysine (DL) to study the effect of mild (45.5°C) heat shock on a variety of mammalian cell lines. It has been previously proposed by Humphries and Lovejoy (1983) that DL selectively partitions into (and diffuses through) membranes whose component molecules have undergone lateral phase separation resulting in the formation of phospholipid domains. Excellent flow cytometric resolution of the DL staining cells from several cell lines was obtained by using bivariate (forward angle light scatter versus DL-fluorescence) analysis. Dye uptake and release data as well as measurement of the octanol: water partition coefficient (7.2) all indicated that the stain was likely associated with the plasma membrane. After heating, all cell lines exhibited a time-dependent increase in the fraction of cells stained by DL. Nearly all of the DL-staining cells were propidium iodide and trypan blue excluding. Exclusion of erythrosin B or inclusion of fluorescein showed a better correlation with colony formation, although neither was found to be as effective as DL in estimating cell killing. A comparison of cell survival curves as measured either by colony formation or by the fraction of cells not stained by DL 24 h after heating indicated a good, though not absolute correlation. These results indicate first that DL may have general usefulness as a stain indicating cell death following heat shock, and second, that DL may have utility as a probe of specific membrane damage induced by heat. Our results are consistent with the hypothesis that membrane lateral phospholipid domain partitioning is associated with hyperthermia-induced cell death in mammalian cells.