A rapid cell membrane permeability test using flourescent dyes and flow cytometry |
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Authors: | Martin Aeschbacher Christoph A. Reinhardt Gerhard Zbinden |
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Affiliation: | (1) Institute of Toxicology, Federal Institute of Technology, University of Zurich, Schwerzenbach, Switzerland;(2) Istituto Cantonale Batteriosierologico, Via Ospedale 6, CH-6904 Lugano, Switzerland;(3) Present address: Istituto Cantonale Batteriosierologico, Via Ospedale 6, CH-6904 Lugano, Switzerland |
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Abstract: | A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS25 concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate |
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Keywords: | cytotoxicity dye retention and exclusion flow cytometry membrane permeability surfactants thymocytes |
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