Generi Biotech - Compounds and Services for Molecular Biology

• Quantitative PCR
  • Sets for Gene Expression
  • qPCR sets for reference genes
  • Custom-Made qPCR Sets
  • Probe Master Mix
  • Standards for qPCR
  • Internal Positive Controls
• Sequencing
  • Standard Sequencing
  • 16S/ITS Sequencing
• Molecular Cloning
  • Custom cloning
  • Plasmid Amplification and Isolation
  • Competent Bacteria
• Cell Culture Applications
  • Recombinant Proteins
  • Recombinant Human SHBG
  • Cytotoxicity Testing
  • Luminescence Solution
  • Mycoplasma Detection
• Biological Identification
  • Cell Line Authentication
  • Paternity Outsourcing

Cytotoxicity Neutral Red Test

The cytotoxicity neutral red test is based on the ability of live cells to uptake and bind neutral red (NR). NR is a positively charged dye that easily diffuses through the cellular membrane of the cells, accumulates in the cellular cytoplasma and is stored in the acidic environment of lysosomes. The principle of the test consists in the fact that NR are able to adsorb and bind only live cells while this ability declines in damaged or dead cells. The amount of accumulated NR is thus directly proportional to the amount of live cells in the cell culture.

Example

Hep2, MCF-7 and HepG2 cells were applied onto the 96-well plate into appropriate medium as follows: 2 x 10⁴ (Hep2), 3 x 10⁴ (MCF-7) or 5 x 10⁴ (HepG2) cells per well. Upon achieving 80% confluence, the medium was replaced and etoposide (ETO) was added in appropriate concentrations, and/or DMSO as a positive control. The cells were incubated with etoposide for 72 hours. Then the neutral red test was performed. Absorbance values reduced by the positive control value are expressed as percentage of viability of the cells related to the negative control sample (uninfluenced cells).

Fig. Etoposide (ETO) cytotoxicity in cell lines Hep2, MCF-7 and HepG2 evaluated using neutral red test. The data were processed in the program GrafPad Prism, version 4 (GraphPad Software, San Diego, CA, USA). IC₅₀ values correspond to etoposide concentration under which the cell viability is equal to 50%. N = 2; n = 3.

References

• Ceckova, M., Z. Vackova, et al. (2008). "Effect of ABCG2 on cytotoxicity of platinum drugs: Interference of EGFP." Toxicol In Vitro 22(8): 1846-52.
• Gramage, E., L. F. Alguacil, et al. (2008). "Pleiotrophin prevents cocaine-induced toxicity in vitro." Eur J Pharmacol 595(1-3): 35-8.
• Chen, P. J., T. Moore, et al. (2008). "Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes." Toxicol In Vitro 22(6): 1476-1483.
• Kitagawa, R. R., L. M. da Fonseca, et al. (2008). "Ascorbic acid potentiates the cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin." Phytochemistry 69(11): 2205-8.
• Repetto, G., A. del Peso, et al. (2008). "Neutral red uptake assay for the estimation of cell viability/cytotoxicity." Nat Protoc 3(7): 1125-31.