Neutral Red (NR) Assay

Abstract

The neutral red (NR) assay is a cell survival chemo sensitivity assay. This experiment is conducted to determine the cytotoxicity of panadol using NR assay. The raw data obtained from the experiment was not really been reproducible as not all the replicates had constant cell growth. However, we still can conclude that panadol, at high concentrations, is toxic to cells. The NR assay has worked in measuring the cytotoxic effect of paracetamol on Vero cells. In addition, NR assay has been less time consuming and easy to conduct. In conclusion, it is advisable not to consume too much panadol at a time (from the instructions on the package of panadol: maximum 8 tablets a day).

Introduction

The neutral red (NR) assay is a cell survival chemo sensitivity assay. This assay is based on the incorporation of NR into the lysosomes of viable cells after being incubation with test agents. NR (3-amino-7-dimethyl-2-methylphenazine hydrochloride) is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion, accumulating intracelluarly in lysosomes, where it binds with anionic sites in the lysosmal matrix. In damaged or dead cells, NR is no longer retained in the cytoplasmic vacuoles and the plasma membrane does not act as a barrier to retain the NR within the cells. Therefore, it is possible to distinguish between viable, damaged or dead cells, which is the basis of this assay.

Principle of Assay

Cells are seeded into individual wells of a 96-well mircotitre tissue culture plate to achieve approximately 80% confluence at the time of the addition of the test agents. After reaching the required confluence, the medium is then replaced with graded dilutions of test agents, filling 4-8 wells per concentration. The NR assay has ability to measure cytotoxic, cytostatic and proliferation effects of the test agents. The plate is incubated for the desired time (ranging from 1 - 6 days) depending on the test agent (for direct acting toxicant requires shorter incubation time as compared to test agents which require metabolic activation). After incubation, the medium is replaced with NR – containing medium and the plate is incubated for an additional 3 hours to allow for the uptake of the dye. The cells are then rapidly washed with a solution of 1% calcium chloride and 0.5% formaldehyde. The damaged or dead cells would lose their ability to retain NR, which is removed during the wash and fixative step. The dye is then extracted from the intact and viable cells with a solution of 1% acetic acid and 50% ethanol. The plate is left to stand at room temperature for 10 – 15mins and agitated for about 30 seconds before measuring the absorbance of the solubilised dye using a spectrophotometer equipped with a 540nm filter. Quantitation of the extracted dye by spectrophotometry can be correlated with viable cell number, both by direct cell counts and by protein determination of cell population.