Trypan blue assay

Cell viability was detected by the MTS assay (Promega, Madison, WI). Briefly, cells were equally seeded into 96-well plates with various concentrations of erastin (0.01, 0.1, 1, 10, and 100 μmol/l). DMSO (Sigma-Aldrich, St. Louis, MO) was used as a negative control. Cell viability was measured using the MTS kit at 24 h. Absorbance was tested at 490 nm using a microplate analyzer (BioTek, Winooski, VT). Cell mortality was detected by a trypan blue assay (Beyotime Biotechnology, Shanghai, China). In brief, cells were stained with trypan blue and counted under an inverted microscope. The blue-stained cells were considered dead, and five random fields per insert were counted. Cell mortality was calculated according to the following formula: cell mortality (%) = number of dead cells/total number of cells × 100.

The Trypan Blue assay (Beyotime Biotechnology, Shanghai, China) was used to evaluate the cell survival rate. The Caco-2 cells were plated in the 12-well plates (1 × 10⁴ cells per well) and incubated for 24 h. Then, the cells were treated with a range of concentrations (0, 0.05, 0.10, 0.15, 0.20 and 0.25 mg/mL) of different C3G nanoliposomes for 24 h. Cells cultured in the free medium were taken as the control. After 24 h, the cells were harvested with 200 μL trypsin-EDTA solution (Solarbio Life Sciences). The mixture of the supernatant and detached cells was centrifuged at 2000 rpm for 2.5 min. The cells were resuspension use the suspension solution. Then, 100 μL cell suepension was added with 100 μL Trypan Blue solution. After 5 min staining, cells were counted using automated cell counter (Bio-Rad, Irvine, CA, USA). Cell survival rate (%) is expressed as percentage of the living cell number/the total cell number [56 (link),57 (link)].