Methyl thiazolyl tetrazolium (mtt)

Ethanol (95%) (BDH laboratory-England), Deionized water, Petroleum ether (BDH laboratory-England), Ethyl acetate (BDH laboratory-England), Rotary evaporator (Heidolph-Germany), Separating funnel. Dulbecco’s modified eagle medium (DMEM) (caisson labs, USA), fetal bovine serum (FBS) (Hyclone, South America) penicillin–streptomycin (Hyclone, US), Phosphate buffered saline (PBS) (Oxoid, England), (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) (Bioworld, USA), Dimethyl sulfoxide (DMSO), syringe filters 0.2 µm, Eppendorf tubes, T-flask 25 cm² (corning, USA), hemocytometer, 96 well plates (corning, USA), petroleum ether dilutions (100 µg/mL, 200 µg/mL and 300 µg/mL), ethyl acetate dilutions (100 µg/mL, 200 µg/mL and 300 µg/mL).

A total of 2 × 10³ HME1 and HME1 (AK + I) were seeded per well in 96-well plates. After 24 h incubation in their appropriate media (DMEM-F12 or DMEM-F12 supplemented with cytokines, inflammatory mediators, and adiponectin), the cells were either treated (or not) in triplicate with doxorubicin overnight. MTT (bioWORLD, Irving, TX, USA) was added, and the cells were incubated for 2–4 h at 37 °C. Then, MTT was removed, and 100 μL of 100% DMSO was added per well. Cells were kept at room temperature, shaking for 5 min; the readings were taken at 570 nm using Corning^® 96-well clear flat bottom polystyrene TC-treated microplates (Costar^®, Kennebunk, ME, USA).

Cell viability was measured using MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay kit (Bioworld, UK). The cells were dispensed (100 μL/well) into each well of 96-well tissue culture plates at a density of 15,000 cells/well. After a 24-h incubation period, the medium in each well was removed, and the cells were treated in triplicate with varying doses of the extracts (initially dissolved in DMSO with final concentration not exceeding 1%). Doxorubicin (5–0.015 mg/ml) was used as a positive control. After 48-h incubation, media were withdrawn from each well, rinsed with phosphate buffer saline (PBS), and replaced with fresh media, followed by adding 20 μL of thiazolyl blue tetrazolium bromide solution, with a 3-h incubation period. One hundred microliters of DMSO was added to each well to terminate the reaction. The cells treated with 1 DMSO were used as negative controls. Microsoft Excel software was used to apply further calculations that estimate the percentage of survival cells and calculate the IC50 values (20 (link)), where OD is optical density:

A375, MCF7, PANC1, MDA-MB-231, Fibro cell lines (5 × 10³ cells per well) and K562 (30 × 10³ cells per well) were seeded in 96-well plates (TPP, Zollstraße, Switzerland). After 24 h, cells were treated with different concentrations of CAP, and liposome loaded with CAP, and without any treatment as negative control; then incubated at 37 °C for 72 h. Then treatments were replaced with 15 μL of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium Bromide (MTT) solution (Bioworld, Visalia, CA, USA) and 100 μL of medium RPMI used for A375, K562, MCF7, and Fibro, while DMEM media used for Panc1 and MDA-MB-231. After incubation for 3 h, the medium was removed, and the cells were mixed with 50 μL of dimethyl sulphoxide (DMSO). The absorbance was measured at a wavelength of 570 nm using a Glomax microplate reader (Promega, Madison, WI, USA).