Cytotoxicity Evaluation of Rh2 in 3T3-L1 Cells

To test whether Rh2 directly kills cells at the selected concentrations, TACS MTT cell proliferation assay (Trevigen, Gaithersburg, MD, USA) was conducted to evaluate cell viability according to the manufactory’s instruction, as we described [23 (link)]. Briefly, 3T3-L1 cells were seeded in 12-well plates (about 20% confluent) with different dosages of Rh2 from 30 μM to 300 μM for 72 h. Cells were then incubated with MTT reagent (100 μL per well) for 4 h and added detergent reagent (500 μL each well) and incubated in the dark for 4 h. The relative cell viability was determined by the absorbance at 570 nm using a Synergy H1 hybrid reader (BioTek Instruments, Inc. Winooski, VT).