A rapid cell membrane permeability test using flourescent dyes and flow cytometry

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.

Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes

“…Similar results were reported using EB and fluorescein with analysis by fluorescence microscopy and flow cytometry (1,14,24). By conventional interpretation the green positive, red negative cells are "viable," while the red positive, green negative cells are classified as "dead.…”

“…7). Cells in Region 1 were uniform in size, as shown by the discrete frequency vs scatter distributions. This is consistent …”

“…The above technique was subsequently adapted to provide rapid flow cytometric analysis of cell membrane permeability (1). Studies with detergents and solvents indicated that the twin probes could be used to ' However, a practical limitation on the use of fluorescein in the present context is imposed by its relatively rapid efflux from a variety of cells with apparently intact cell membranes (6,8,19,22,23).…”

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…36,37 The accumulation or release of fluorescein after hydrolysis of FDA is dependent on the cell membrane permeability and integrity. [37][38][39][40] Hepatocyte polarity in culture has been observed by examining the secretion of fluorescein from hepatocytes through gap junctions into intercellular clefts. 38,41 When paracellular junction integrity is compromised, intercellular gap junction communication is disrupted, and fluorescein is able to passively diffuse out of the cells through the gap junctions.…”

Multiparameter flow cytometric analysis of a novel cytotoxin (Factor 2) induced tumor cell membrane permeability

“…An earlier technique for cell membrane damage combined fluorescein and ethidium bromide together as twin fluorescent probes for flow cytometric analysis of cell 'Address reprint requests to Dr. A. Karpas, Department of Haematology, University of Cambridge, MRC Centre, Cambridge, CB2 2QH, England. membrane permeability (1). However, a practical limitation on the use of fluorescein is imposed by its relatively rapid efflux from a variety of cells with apparently intact cell membranes (4).…”