Neutral Red Uptake Assay

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 and accumulates in the lysosomes. Dying cells have altered membrane properties and therefore they cannot take up neutral red (NR) anymore. Consequently, living cells can be distinguished from dead or dying cells based on their different NR uptake. After the cells have been exposed to 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. Based on the absorption values, viability curves can be established to determine the concentration of test substance that is responsible for 50 % inhibition of NRU (IC50 or NI50).