Mtt assay kit

Cell viability was assessed using an MTT assay kit (ATCC, Manassas, VA, USA) in accordance with the manufacturer’s instructions. In brief, cells were cultured in 96-well microplates at a density of 2 × 10⁴ cells/well. After treatments with MuV and chemical inhibitors, the cells were incubated with 10 μl of MTT solution. After 2 h, 100 μl of the detergent reagent, which was included in kit, was added to each well. The absorbance at 570 nm was determined with a microplate reader (BioTek, Winooski, VT, USA). The percentage of the absorbance value versus the control value represents cell viability.

Cell viability was determined using an MTT assay kit (ATCC, Manassas, VA) or LDH assay (Thermo Scientific, Waltham, MA). Briefly, cells were cultured in 96-well plates at a density of 2,000 cells/well and exposed to normoxia or hypoxia for 3 or 6 days. For MTT assays, 10 µL of MTT reagent was added to the cells. After 4 hrs of incubation, 100 µL of detergent reagent was added. Plates were maintained at room temperature in the dark for 2 hrs, and the absorbance values were subsequently recorded at 570 nm. For LDH assay, 50 µL of the supernatant was transferred to new 96-well plates. Then, 50 µL of the reaction mixture (combination of substrate and assay buffer) was added to the supernatant, and plates were incubated at room temperature in the dark. After 30 min, 50 µL of stop solution was added, and the absorbance was measured at 490 nm and 680 nm. Absorbance values at 680 nm (background) were subtracted from absorbance values at 490 nm to obtain LDH activity.

Cell proliferation was examined following instructions of MTT assay kit (ATCC, Manassas, VA). Briefly, OVCAR3 or OVCAR8 cells (3,000/well) were plated into 96-well plates and treated with different doses of FAK PROTAC, VS6063 or vehicle for various time points, and then MTT reagent (5mg/mL) was added to each well. After a 4-hour incubation, the media/MTT was aspirated and 100 μL DMSO was added to each well. The plate was incubated for 20 min and the absorbance was measured at single-wavelength mode (490 nm) using a Multiskan MK3 Microplate Reader (Thermo Fisher Scientific). BrdU proliferation assay was performed by labelling OC cells for 12 h with 10 µM BrdU and cells were fixed with 3.7% formalin and cells were washed and incubated with BrdU antibody (Santa Cruz, Cat.No. SC-32323) for 24h at 4°C and then fluorescent labelled secondary antibody Alexa 488 (Thermo Fisher Scientific. Cat. No. A32723) for 1h at room temperature. Cell proliferation was detected by counting BrdU labelled cells and DAPI labelled cell nuclei from four different fields under fluorescent microscopy.

Cell viability was assessed using an MTT Assay Kit (ATCC, Manassas, VA, USA) in accordance with the manufacturer’s instructions. In brief, cells were cultured in 96-well microplates at a density of 2 × 10⁴ cells/well. After treatments with MuV and 3-MA, cells were incubated with 10 µL of MTT solution for 2 h. Then, 100 µL of detergent reagent, which was included in the kit, was added to each well. Absorbance at 570 nm was determined with a microplate reader (BioTek, Winooski, VT, USA). The percentage of the absorbance value versus the control value represents cell viability.

In a 6-well plate, MSCs were incubated with brefeldin A (Sigma-Aldrich) at a final concentration of 50, 10, 5, and 1 µg/ml for 24 hours. Supernatants were collected, and then MSCs were washed 3 times using PBS. Fresh medium was replaced, and cells continued to culture up to 3 days. Supernatants were collected at different time points for subsequent human IL-6 measurements. To measure the proliferation, BFA-treated cells were reseeded in a 96-well flat bottom plate and cultured with fresh medium for 72 hours. Cell proliferation was measured using a MTT assay kit (ATCC) following the supplier’s recommended procedures.