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Neutral red uptake assay for the estimation of cell viability/cytotoxicity


The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

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Figure 1: Cultured cells stained with neutral red.
Figure 2: Relationship between the time of incubation with neutral red and the absorbance at 540 nm after the assay of neutral red.
Figure 3: Relationship bewteen the number of cells and the absorbance at 540 nm after the assay of neutral red.
Figure 4: Neutral red cytotoxicity test.


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This work was partially supported by a grant from the Spanish Ministry of Education and Science and FEDER, project CTM 2006-06618.

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Correspondence to Guillermo Repetto.

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Repetto, G., del Peso, A. & Zurita, J. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3, 1125–1131 (2008).

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