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NHEK Neutral Red Uptake Assay for Ocular Irritation

The Normal Human Epidermal Keratinocytes (NHEKs) Cytotoxicity Assay using a Neutral Red Uptake (NRU) viability endpoint is a 96-well cytotoxicity assay to assess the toxicity potential of a test material. The assay utilizes primary NHEK cells and may be used to predict either human lethal serum concentrations or in vivo rodent LD50 starting doses for acute oral systemic toxicity. Cytotoxicity caused by the test article is measured by a concentration-dependent reduction in neutral red uptake (NRU) by the cells after exposure to a test material.

Healthy mammalian cells, when maintained in culture, continuously proliferate. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in a reduction in viable cells relative to untreated controls. The viability of the cells can be assessed using a neutral red uptake endpoint. A decrease in the uptake of neutral red dye in treated cell cultures following a test chemical exposure is used to determine relative toxicity.

For specific assay procedures, please see Step-by-Step.

Assay Design: Quick Facts

Assay Model: NHEK cells seeded in a 96-well plate

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

Each assay includes a positive and negative control. 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.