A cyanine dye distinguishes between cycling and non-cycling fibroblasts

Abstract

Cellular proliferative activity has previously been determined by measuring the incorporation of radiolabelled nucleotides or by visual inspection of cellular morphology. Although two flow cytometric methods^1,2 have recently been developed which can distinguish cycling from non-cycling cells, both have serious disadvantages. One method requires uptake of a substantial amount of BUdR¹, limiting its usefulness for in vitro systems. The other method utilizes RNA/DNA content differences² but its successful application has proved cell-type dependent. We have now used the findings that the cell membrane is more highly polarized in resting than in proliferating cells³ and that cyanine dyes carrying a delocalized positive charge enter live cells to an extent that depends on the cell membrane potential⁴, to develop a method of distinguishing between cycling and non-cycling cells. The greater the membrane polarization, the greater is the concentration of dye within the cell. At high concentrations, the dye molecules aggregate and their fluorescence is quenched⁵. Thus, for a given external dye concentration, cells of different membrane potential would accumulate different amounts of fluorescent (non-aggregated) dye. Using fibroblasts in culture conditions chosen to provide various models of cycling and non-cycling cells, we found that fluorescence intensity with the dye 3,3'-diheptyloxycarbocyanine (Di-O-C₇(3))⁶ was consistently greater in the former than the latter.