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3T3 Neutral Red Uptake Phototoxicity


The 3T3 Neutral Red Uptake (NRU) Phototoxicity assay is a 96-well cytotoxicity-based assay that utilizes normal BALB/c 3T3 mouse fibroblasts to measure the concentration-dependent reduction in neutral red uptake by the cells after exposure to a test material either in the presence or absence of UVA light. Duplicate 96-well monolayers of 3T3 fibroblasts are exposed to serial dilutions of a test material. One of the plates is exposed to 5 J/cm2 UVA while the other plate is kept in the dark. To assess viability, the neutral red uptake (NRU) by cells exposed to the test chemical in the presence of UVA exposure is compared to the NRU by cells exposed to the test chemical in the absence of UVA exposure.

Neutral Red (NR) is a weak cationic dye that readily diffuses through cell membranes and accumulates in cellular lysosomes. Once in the acidic environment of the lysosome, the NR is oxidized, becoming positively charged and trapped within the lysosome. Unless the cell or lysosome is damaged, the red dye remains trapped. Alterations to the cell membrane (caused by toxicity of the test material) are generally irreversible, resulting in the loss of the NR from the lysosome. For specific assay procedures, please see Step-by-Step.

Assay Design: Quick Facts

Assay Model: 3T3 cells seeded in duplicate 96-well plates

Endpoints: calculated using Phototox 2.0 software (supplied by ZEBET)

  1. IC50 (the concentration of test material that causes a 50% decrease in viability, relative to the solvent control)

  2. PIF (Photo-Irritation-Factor) compares the IC50 of treated cells in the presence of UVA exposure to the IC50 of treated cells in the absence of UVA exposure.

  3. MPE (Mean Photo Effect) compares the response curve of treated cells in the presence of UVA exposure to the response curve of treated cells in the absence of UVA exposure.

For more information about testing your materials using this assay, please see Applications. Specialized protocols may be prepared as requested through consultation with an IIVS Study Director

3T3 NRU Phototoxicity Assay
3T3 NRU Phototoxicity Results