In vitro cell viability assay

Procedure for in vitro cytotoxicity assay was adapted from [10]. Five hundred cells (at a concentration of 50,000 cells/ml of media) were seeded in 96-well plates (100 μl per well) and incubated for a day to facilitate attachment to the bottom. The next day, old media was aspirated and the cells were incubated with 100 μl of different formulations and 100 μl of fresh media for 3 days (200 μl total volume). 100 μl of PBS was added as a negative control post 3-day incubation, the supernatant was aspirated and cells were incubated with 100 μl of fresh media and 11 μl of MTT solution (5 mg/ml in PBS) for 3.5 h at 37°C and 5% CO₂. The supernatant was then aspirated and MTT formazan crystals were then dissolved in 100 μl of DMSO per well, and absorbance was measured using Tecan Infinite F200 plate reader at 550 nm. Cell viability was proportional to (absorbance reading of well–absorbance of blank DMSO). Curve fitting and data analysis were performed using OriginPro 8 software.