Trypan blue solution

Methoxy poly(ethylene glycol) 5000 (mPEG), D,L-lactide, cholesteryl chloroformate, curcumin, tetrahydrofuran (THF), DAPI 4′,6-diamidino-2-phenylindole dihydrochloride (4,6- diamidino-2-phenylindole), and para-formaldehyde were purchased from Sigma-Aldrich Chemicals (Germany). Thiazoyl blue tetrazolium bromide (MTT), fluoromount-G and trypan blue solution were obtained from Himedia (Mumbai, India). Dialysis membrane was purchased from Spectrum Laboratories, Inc., Piscataway Township, NJ.
For cell culture, Dulbecco’s modified eagle’s medium (DMEM), growth medium RPMI-1640, penicillin–streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from Himedia (Mumbai, India). All reagents were used as received and the solvents were purified according to the general procedures.

T. musilii methanolic extract was tested against human lung (A549), breast (MCF-7), and colon (HCT-116) cancer cells. For the experiment, cell lines were cultured upon 80% confluence, and 0.4% Trypan Blue solution (Hi-Media, India) (0.4%) was used to stain the cells. Then, cells were treated for 48 h with various T. musilii concentrations ranging from 100 μg/mL to 500 μg/mL. Afterward, 200 μL of medium containing 10% MTT reagent was added to each well to obtain a final concentration of 0.5 mg/mL, and the plates were incubated for a further 3 h at 37 °C with 5% CO₂ atmosphere. After removing the medium, 100 μL of DMSO was added to dissolve the formed formazan crystals. The absorbance was measured at 570 nm and 630 nm. The percentage growth inhibition was calculated after subtracting the background and the blank, and the concentration of the test drug needed to inhibit cell growth by 50% (IC₅₀) was calculated from the dose–response curve for the respective cell line [37 (link),38 (link)].