Neutral Red Uptake Assay in C3a cells

Commonly used acronym: NRU

Scope of the method

Alternative method relates to
  • Human health
Alternative method is situated in
  • Basic Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
C3a cells (Hepatocellular Carcinoma Cells, the C3A cell line is a clonal derivative of Hep G2 cells )

Description

Method keywords
  • neutral red
  • IC50
  • NI50
  • cell viability test
Scientific area keywords
  • in vitro cytotoxicity
  • acute toxicity
Method description

The neutral red uptake (NRU) assay provides a quantitative measurement of the number of viable cells. The test is based on the ability of living cells to take up and bind neutral red (NR), a dye which easily penetrates cell membranes via non-ionic diffusion. It accumulates in the lysosomes. Dying cells have altered membrane properties and therefore they cannot take up neutral red (NR) anymore. Living cells can therefore be distinguished from dead or dying cells based on their different NR uptake. After the cells have been allowed to incorporate the dye for three hours they are briefly washed with PBS. The incorporated dye is then liberated from the cells in an acidified ethanol solution. Released neutral red (proportional to the amount of Viable cells) is measured at OD 540 nm (OD 620nm as reference). Measured OD 540 nm of unexposed cells is set to 100% viability. According to this, after exposure with test substance cell viability or viability curves can be established which enables determination of the concentration of test substance that is responsible for 50 % inhibition of NRU (IC50 or NI50).

Lab equipment

Spectrofotometer

Method status
  • History of use

Pros, cons & Future potential

Advantages
  • Very sensitive and readily quantifiable.
  • Simple, fast, accurate and yields reproducible results
  • Cheap
Challenges
  • Crystal formation neutral red dye (critical step in the protocol)
  • Possible Interference with colored compounds
Future & Other applications

Use of the NRU assay with non adherent cells (like TK6 cells)

References, associated documents and other information

References

Borenfreund, E. and Puerner, J. [1985] Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24:119-124

Repetto G, Del Peso A, Zurita JL. Neutral red uptake assayfor the estimation of cell viability/cytotoxicity. Nat Prot. 2008;3:1125–31

Ndhlala AR, Anthonissen R, Stafford GI, Finnie JF, Verschaeve L, Van Staden J. In vitro cytotoxic and mutagenic evaluation of thirteen commercial herbal mixtures sold in KwaZulu-Natal, South Africa. S Afr J Bot. 2010;76:132–8

Associated documents
NRU-revieuw.pdf In vitro cytotoxic and mutagenic evaluation of thirteen commercial herbal mixtures sold in KwaZulu-Natal, South Africa.pdf Toxicity determined in vitro by morphological alterations and neutral red absorption..pdf

Contact person

Roel Anthonissen

Organisations

Sciensano
Chemical and physical health risks
Risk and health impact assessment
Belgium