Cell Viability Assay: Neutral Red Uptake Assay Protocol

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About Neutral Red Cell Viability Assay

The NRU cytotoxicity assay or Neutral red uptake assay procedure is a cell survival or cell viability chemosensitivity assay based on the ability of live cells to incorporate and bind neutral red (NR), a supravital dye.

Principle of Neutral Red Cell Viability Assay

NR is a weak cationic dye that rapidly enters into cells via cell plasma membranes by non-ionic diffusion and accumulates intracellularly in lysosomes.

Cell surface alterations or the sensitive lysosomal membrane lead to lysosomal fragility and other modifications that gradually become irreversible.

These changes brought about by the action of any toxic compound result in a decreased uptake and binding of NR.

It is thus possible to distinguish between dead cell, viable cells, damaged cells, which is the basis of the NRU cytotoxicity assay or Neutral red uptake assay.

Healthy and viable mammalian cells, when maintained in culture conditions, continuously proliferate and divide over time.

Any chemical compound that will interfere with this process and result in a reduction of the growth rate due to toxic nature of compound as reflected by cell number or cell death.

Cytotoxicity is expressed as a concentration dependent reduction of the uptake of the NR after chemical exposure thus providing a sensitive, integrated signal of both cell integrity and growth inhibition.

Neutral Red Cell Viability Assay

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Neutral Red Cell Viability Assay Requirement


1. Secondary cell line

2. Growth medium with 10% FCS; Growth medium with 5% FCS

3. Trypsin (0.25%) + EDTA, (1 mM) in PBS

4. Multiwell plates (96 well), Reservoir for multichannel pipette.

5. Pipettor tips, in an autoclavable tip box

6. Universal containers or tubes

7. Cytotoxic drug, 70% IPA

8. Neutral red stock solution (3.3 mg/ml in water)

9. Neutral red medium (1:100 dilution of neutral red stock in medium)

10. Neutral red desorbing medium(50% ethanol, 1% glacial acetic acid)


11. Plastic box (clear polystyrene, to hold plates)

12. Multichannel pipettor, neuber’s chamber

13. Dimethyl sulfoxide (DMSO)

14. ELISA plate reader

Neutral Red Cell Viability Assay Procedure

Cells are plated in 96 well plate at a concentration of 1×104 cells / well.

The medium used is RPMI-1640 medium supplemented with10% FBS (Fetal Bovine Serum).

The plate is incubated for 24 hr at 37 C in CO2 incubator with 5% CO2.

The dilutions of the drugs are prepared in RPMI-1640 medium supplemented with 5% FBS.

After 24 hr of seeding, medium is removed and cells are treated with different concentrations of drug.

Treatment is done in 96 well plates and is same as given for MTT assay.

150 μl of drug containing medium is added per well and incubated for 72 hr at 37oC in CO2 incubator.

Control wells are treated with drug free medium. DMSO control wells are treated with medium containing DSMO (as calculated by the dilutions used for the assay)

After the incubation period of 72 hr, the medium is removed.

150 μl of Neutral red medium is added to each well and the plate is incubated at 37C in CO2 incubator.

After the incubation period of 3 hr, medium is removed and cells are washed with 1X PBS and 100 μl of neutral red desorbing solution is added.

Record optical density (O.D.) at 540/570 nm using ELISA plate reader.

Cell viability is calculated as a ratio of control and plotted against log concentration of drug to calculate the IC50.

Analysis of Neutral Red Cell Viability Assay

Plot a graph of the absorbance (y-axis), considering cells without drug as 100% against the concentration of drug (x-axis).

Calculate the LC50 as the drug concentration that is required to reduce the absorbance to half that of the control.

Neutral Red Cell Viability Assay Citations:


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