A SIMPLE QUANTITATIVE PROCEDURE USING MONOLAYER CULTURES FOR CYTOTOXICITY ASSAYS (HTD/NR-90)

A SIMPLE QUANTITATIVE PROCEDURE USING MONOLAYER CULTURES FOR CYTOTOXICITY ASSAYS (HTD/NR-90)

Vol. 9, No. 1, 1984 | Ellen Borenfreund and James A. Puerner
The article describes a sensitive quantitative procedure for assessing cytotoxicity in monolayer cultures using a two-component test. The first component involves microscopic screening to detect morphological alterations after an experimental protocol, while the second component quantifies surviving cells by incubating with the supravital dye neutral red and analyzing the dye extracted from the lysosomes of viable cells. Both assays are standardized for use with 96-well microtiter plates and can be read automatically with a Dynatech spectrophotometric microplate reader. The method is particularly advantageous for monolayer cultures, eliminating the need for trypsinization and the trypan blue exclusion test. The procedure is detailed, including the preparation of cell cultures, the application of test agents, and the scoring of cytotoxic effects. The highest tolerated dose (HTD) and neutral red assays are described, with the latter involving washing the cultures and measuring the dye incorporation. The discussion highlights the rapid, reliable, and reproducible nature of the assay, making it suitable for screening cytotoxic agents.The article describes a sensitive quantitative procedure for assessing cytotoxicity in monolayer cultures using a two-component test. The first component involves microscopic screening to detect morphological alterations after an experimental protocol, while the second component quantifies surviving cells by incubating with the supravital dye neutral red and analyzing the dye extracted from the lysosomes of viable cells. Both assays are standardized for use with 96-well microtiter plates and can be read automatically with a Dynatech spectrophotometric microplate reader. The method is particularly advantageous for monolayer cultures, eliminating the need for trypsinization and the trypan blue exclusion test. The procedure is detailed, including the preparation of cell cultures, the application of test agents, and the scoring of cytotoxic effects. The highest tolerated dose (HTD) and neutral red assays are described, with the latter involving washing the cultures and measuring the dye incorporation. The discussion highlights the rapid, reliable, and reproducible nature of the assay, making it suitable for screening cytotoxic agents.
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