Cell counting kit 8 (cck8)

To assess the completeness of total gastric vagotomy and SAP vagal deafferentation, a CCK-8–induced food intake suppression test was performed as previously described (65 (link)). Mice were deprived of food for 20 hours from 17:00 to 13:00 hours the next day. During the testing, CCK-8 (8 μg/kg; MilliporeSigma) was injected i.p. 15 minutes before the food-intake test. For the food-intake test, mice were allowed ad libitum feeding for 2 hours, and weight of food pellets was recorded before and after consumption. Food intake percentage was normalized to body weight.

BMMs cultures were raised with M-CSF for 3 days. Cell proliferation was measured using the Cell Counting kit-8 (Millipore Sigma) as per manufacturer’s instructions [33 (link)]. For detection of cell death, BMMs were cultured to raise pre-osteoclasts with M-CSF and RANKL for 3 days in a 48-well plate. Pre-osteoclasts were subjected to cytokine and serum starvation for 4 h followed by assaying for apoptosis using the Cell Death Detection ELISA^PLUS kit (catalog No. 11774425001, Roche, Basel, Switzerland) as per manufacturer’s instructions.

The number of viable cells over time was determined similarly. BvAdMSCs were seeded into 12-well plates at a density of 3000 cells/cm². Each day, starting from d 1 until d 6, the cells were trypsinized, neutralized with double the amount of culture maintenance medium, and scraped until the cells were detached from the flask. The cell suspension was collected in microcentrifuge tubes. Then, the cells were centrifuged at 1300 rpm for 5 min to obtain a cell pellet. The supernatant was discarded, and the cells were resuspended in 1 mL of cell culture maintenance medium. Cell suspension from each well (100 µL) was added to a 96-well plate in duplicates. The 96-well plate was incubated at 37 degrees Celsius and 5% CO₂ for 6 h before adding the Cell Counting Kit-8 (cat.no. 96992, Millipore Sigma, Saint Louis, MO, USA) solution. The plate was then incubated for 2 additional hours. A plate reader was used to detect the absorbance at 450 nm. The cell number was determined using a standard curve made by known numbers of viable cells and their respective absorbances.

Cells were suspended in medium and counted using the Countess Automated Cell Counter (Invitrogen). About 3,000 cells were seeded to each well of the 96-well plates. After the cells adhered to the plate, 10 μL of the Cell Counting Kit-8 (MilliporeSigma) solution was added to each well and incubated for 2 hours in the 37°C CO₂ incubator, followed by measuring of the absorbance at 450 nm using the Synergy HTX Multi-Mode Microplate Reader (BioTek). Absorbance was measured for 3 additional days to calculate the relative cell proliferation. To measure the proliferation of cells with siRNA knockdown, cells were transfected with siRNAs using Lipofectamine RNAiMax (Invitrogen) at least 6 hours before trypsinization for cell counting and seeding into 96-well plates. At least 5 wells were measured to calculate the average absorbance for each cell line at each time point.