Celltiter glo 2.0 cell viability assay kit

5 × 10³ cells were plated in a 96-well plate. After 96 h, cell viability was measured using CellTiter-Glo 2.0 Cell Viability Assay kit (Promega; Madison, WI, USA; Cat# G9241) according to the manufacturer’s protocol. The assay was performed in triplicates to ensure proper statistical analyses.

To determine MSC cytotoxicity, cells were seeded in 96-well plates (BD Falcon, Durham, NC, USA) at a density of 400 cells/mm² for HEPG2 and Hep3B cells and 300 cells/mm² for Huh7 cells 24 h before MSC treatment. For the determination of the half-maximal inhibitory concentration (IC₅₀), MSC was serially diluted to ten different concentrations. The ATP content of the lysed cells, which is proportional to the number of viable cells, was measured after 48 and 72 h using the luminescence-based CellTiter-Glo^® 2.0 cell viability assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions with an FLx100 Luminometer (CLARIOSTAR^®, Ortenbery, Germany).

The crypt was isolated from the Apc^Min/+ mouse intestine using ethylenediaminetetraacetic acid (EDTA). A total of 500 isolated crypts were mixed with 50 μL of Matrigel (BD Bioscience, San Jose, CA, United States) and plated in 24-well plates. After Matrigel polymerization, 500 μL of IntestiCult Organoid Growth Medium (STEMCELL Technologies, Vancouver, Canada) was added on the top for organoid culture. Organoids were maintained at 37 °C in a Forma incubator (Thermo Fisher Scientific, Waltham, MA, United States) containing 5% CO2 and 95% humidity. Organoids were treated with different concentrations of triptolide for 72 h. ATP levels were measured using the Cell-Titer Glo2.0 Cell Viability Assay Kit (Promega, Madison, WI, United States) according to the manufacturer’s instructions.

Cellular ATP content was measured using a CellTiter-Glo^® 2.0 Cell Viability Assay kit (Promega, Madison, WI, USA). Briefly, myotubes were washed after treatment with cold DPBS, homogenized in ATP sample buffer, and centrifuged at 13,000 rpm for 20 min. An amount of 50 μL of supernatant was added into each well of a 96-well plate, followed by the addition of ATP reaction mix solution at 25 °C for 30 min. The absorbance (OD 570 nm) was assessed by a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer).

Cells were seeded in quadruplicates at a density of 2,500–5000 cells in 100 μl DMEM per well of the white walled 96-well plates (Corning/Costar, 3917). Next day, the medium was aspirated, each well was washed with 200 μl PBS after which treatment medium was introduced. At the end of the experiment duration (48 or 72 h), relative proliferation was determined with CellTiter-Glo 2.0 Cell Viability Assay Kit (Promega, G9243) and the luminescence quantified using a SpectraMax M3 Microplate Reader.

To determine the glutamine and glutamate level in cells, we used Glutamine/Glutamate-Glo assay kit (Promega). Extracts from 20,000 cells were used for each wells. For ATP assay, we used CellTiter-Glo 2.0 cell viability assay kit (Promega). 10,000 cells were seeded onto white 96-well plate and equilibrated at room temperature for 30 min 100 μL of detection reagent were added and incubated for 10 min. Luciferase signals from both assays were measured by Synergy LX multi-mode microplate reader (BioTek).

Cell viability and caspase-3/7 activities were evaluated using the CellTiter-Glo® 2.0 Cell Viability Assay kit (Promega, Madison, WI, USA) and Caspase-Glo® 3/7 3D Assay kit (Promega, Madison, WI, USA), respectively, according to the manufacturer’s instructions. H1975, PC9, H3122, and HC827 cells were plated at a density of 1.0 × 10⁴ cells/well in 50 µL of complete culture medium into 96-well plates. Then, 50 µL of each drug was added at the indicated final concentration (i.e., 0.1, 1.0, 10, and 100 µM). After 6 h, 100 µL of the Viability reagent were added to all wells. The samples were then mixed with an orbital shaker for 2 minutes and incubated at room temperature for 10 min. Luminescence was measured for viability using a plate reader (Enspire; PerkinElmer, Waltham, MA, USA). Next, H1975, PC9, H3122, and HC827 cells were plated at a density of 5.0 × 10³ cells/well in 10 µL of complete culture medium into 384-well plates. Then, 10 µL of each of the drugs were added at the specified final concentration. After 6 h, 20 µL of the Viability reagent were added to all wells. The samples were then mixed with an orbital shaker at 500 rpm for 30 s, and incubated at room temperature for 30 min. The caspase activation was determined by measuring the luminescence with an Enspire instrument.