Neutral red is a vital dye, is used as an indicator and biological stain.

Cell Neutral red uptake assay
Procedure:
1.Cells were cultured in a 96-well plate under normal conditions until they reached a confluency of 70-80%.
2.The neutral red working solution (prepared the day before the neutral red uptake assay and incubated overnight in a 37°C cell culture incubator) was centrifuged at 4,000 g for 5 minutes at room temperature (20-30°C) to remove any precipitate.
3.200 μL PBS was added to each well of the 96-well plate to rinse the cells.
4.100 μL of neutral red working solution was added to each well using a dispenser.
5.Incubated in a 37°C incubator (5% CO2) for 2-4 hours. Note: For RD and MRC-5 cell lines, the optimal incubation time is 2 hours, and longer incubation times will result in significant sensitivity loss. For Vero E6 and A549-hACE2 cell lines, the incubation time can be up to 4 hours without affecting the detection sensitivity;
6. Add 200 μL PBS to each well to wash the cells and remove the neutral red working solution.
7. Aspirate the wash buffer and gently pour the remaining buffer on a paper towel or let it air dry. Note: The wells need to be completely dry before adding neutral red destaining solution.
8. Add 100 μL of neutral red destaining solution (usually 50% ethanol, 1% acetic acid or DMSO) to each well. Shake the plate quickly at 200 rpm on a microplate shaker for 15 minutes to allow the destaining solution to completely extract neutral red from the cells and form a uniform solution.
9. Measure the absorbance of the neutral red extract at 540 nm using a spectrophotometer.