Spectrostar omega microplate reader

An analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The p38α active protein (10 ng, Promega, Madison, WI, USA) was assayed in a kinase reaction buffer with 500 ng c-MYC human recombinant protein (Abcam, Cambridge, MA, USA), 150 µM ATP, and varying concentrations (0.01, 0.1, and 1 µM) of ralimetinib (LY22228820). A total of 500 ng of p38 peptide substrate was used as a control. The generated luminescence was measured using a SPECTROstar Omega microplate reader (BMG Labtech, Offenburg, Germany).

The cytotoxicity of MnBuOE per se in both NSCLC cells was evaluated by the CV staining assay as previously described [38 (link)]. Briefly, 3 × 10³ cells/well were seeded in 200 μL of complete medium in 96-well plates and incubated for 24 h at 37 °C under 5% CO₂ atmosphere. After incubation, the culture medium was changed and cells were exposed to different MnBuOE concentrations (0.5–200 μM) for 72 h in order to assess a concentration–response profile. Cisplatin (50 μM) was used as a positive control for both cell lines. After the 72 h incubation period, PBS was used to wash the cells in order to remove non-adherent cells. The viable cells were fixed with ice-cooled EtOH and stained with 0.1% CV for 15 and 5 min, respectively. The excess of the dye was then removed with tap water and stained cells were resuspended with a solution of 96% EtOH/1% acetic acid. Absorbance was measured at 595 nm with a SPECTROstar OMEGA microplate reader (BMG Labtech, Offenburg, Germany). Absorbance values of the control cells were defined as 100% cell viability. Five independent experiments were performed and six replicates were used for each condition in each independent experiment. The Motic AE2000 Inverted Phase Contrast Microscope was used to perform image acquisition with a 40× objective.

FACS counting-based killing assay was performed as previously described with some modification [3 (link)]. Briefly, CAR-Ms or control were plated in 48-well tissue culture plates one day before the cytotoxicity assay, then CellTrace Violet-labeled target tumor cells were added to the plate. After 24 h or 48 h coculture, the remaining number of target tumor cells remaining was analyzed by FACS as follows: 10,000 CountBright^TM absolute counting beads (Thermo Fisher) were added to the well immediately prior to reading and the cell-counting bead mixture was harvested by pipetting up and down. Percentage cytotoxicity was calculated as: % cytotoxicity = [(1 − (the remaining number of target cells from treated groups/the number of target cells alone)] × 100.
Luciferase-base killing assay was performed as previously described [5 (link)]. Briefly, target tumor cells expressing luciferase were cultured with effector cells for 48 h in a white-walled 96-well plate. D-luciferin (150 μg/mL) was added 10 min prior to the bioluminescence reading in a SPECTROstar Omega microplate reader (BMG Labtech, Ortenberg, Baden-Württemberg, Germany). Percent specific lysis was calculated on the basis of luciferase signal (total flux) relative to tumor alone, using the following formula: % specific lysis = [(Sample signal − Tumor alone signal)/(Background signal − Tumor alone signal)] × 100.

Conventional MTT assay was used to evaluate the cytotoxic effect of Schisandra. AMs (1 × 10⁵ cells/well) were seeded and plated into 96-well plates, then incubated in serum-free medium for 2 h. After appropriate treatment according to the experimental grouping, the cells were incubated with Schisandra medicated serum (10%, 20%, 30%, and 40%, v/v) for 48 h. Subsequently, each well was added to 20 μl of MTT (5 mg/ml, Solarbio, China), and the cells were incubated for an additional 4 h. Then, the supernatants were removed, and the formazan crystals were dissolved in DMSO (100 μl, Sigma, USA). Finally, absorbance was measured at 570 nm using a SPECTROstar Omega Microplate Reader (BMG Labtech, Germany).