**4.11 Neutral red assay**

The neutral red dye easily penetrates nonionic cell membranes and accumulates in lysosomes. The structural integrity of these lysosomes serves as an indicator of cell viability, which is the foundation of the neutral red uptake assay. This method can quantitatively measure live cells [40]. The neutral red assay technique is based on the degree of absorption and binding of the dye by living cells [41]. This cell viability assay aids in the in vitro evaluation of biomaterials. The underlying principle is that dying cells, due to altered membrane properties, can no longer take up neutral red. As a result, living cells can be distinguished from dead or dying cells based on differences in neutral red uptake. After being exposed to the dye for 3 hours, the cells are briefly rinsed with a phosphate buffer solution. Cells are seeded on a 96-well plate and then exposed to test material or control substances in a nutritive cell culture medium for 24 hours. Live cells take up neutral red into their lysosomes after 24 hours of exposure, but as cells begin to die, their ability to incorporate neutral red decreases. The cells are subsequently treated with an acidified ethanol solution to release the incorporated dye. Neutral red that has been released is measured at 540 nm and correlated to the number of viable cells. The viability of unexposed cells measured at 540 nm is set at 100%. The lysosomal capacity for dye incorporation, the foundation of the neutral red dye assay, can be employed to differentiate between living, injured, and dead cells. Viability curves can be generated based on absorption data to determine the concentration of the test chemical needed to inhibit neutral red dye uptake by 50%. Lysosomal swelling agents have been demonstrated to cause an increase in neutral red uptake, potentially leading to an underestimation of cytotoxicity [42]. However, the neutral red assay can produce false-positive or false-negative results. The neutral red assay is more affordable and sensitive than many other cytotoxicity tests [43]. However, being a sensitive test, it must be completed immediately once initiated, usually within 3 hours after the cells have been treated with the dye [41]. This assay does not require unstable reagents like tests using tetrazolium salts. Another limitation is that the absorbance readings' accuracy is affected by the visible needle-like crystal precipitates of the neutral red dye [44].
