Assaying Cellular Viability Using the Neutral Red Uptake Assay

Commonly used acronym: NRU

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
  • Education and training
  • Regulatory use - Routine production
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Specify the type of cells/tissues/organs
Hepatic cell lines such as HepG2, HepaRG. Other cell lines also possible, e.g. 3T3 mouse fibroblasts.


Method keywords
  • cellviability
  • toxicity
  • acute toxicity
  • neutral red uptake
  • HepG2
Scientific area keywords
  • in vitro toxicity
  • viability study
  • hepatic toxicity
  • basal toxicity
Method description

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications.

Lab equipment
  • Incubator (37 ± 1 °C, 90 ± 5% humidity, 5.0 ± 1% CO2/air) ;
  • Laminar flow / clean bench / cabinet (standard: “biological hazard”) ;
  • Water bath (37 ± 1 °C) ;
  • Inverse-phase contrast microscope ;
  • Laboratory balance ;
  • 96-Well plate spectrophotometer (i.e., plate reader) equipped with 540 ± 10 nm filter ;
  • Shaker for microtiter plates ;
  • Cell counter or hemocytometer ;
  • Pipettes, pipettors (multichannel and single channel; multichannel repeater pipette) ;
  • 96-Well flat-bottom tissue culture microtiter plates ;
  • Multichannel reagent reservoir ;
  • Vortex mixer.
Method status
  • Published in peer reviewed journal
  • Validated by an external party (e.g. OECD, EURL ECVAM,…)

Pros, cons & Future potential

  • Fast ;
  • Accurate;
  • Cheap.

Relatively easy to perform.

References, associated documents and other information


Ates, Gamze, Tamara Vanhaecke, Vera Rogiers, and Robim M. Rodrigues. "Assaying Cellular Viability Using the Neutral Red Uptake Assay." Cell Viability Assays: Methods and Protocols (2017): 19-26

Associated documents
NRU Book chapter.pdf

Contact person

Robim Rodrigues


Vrije Universiteit Brussel (VUB)
Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology