Cell counting kit 8 cck 8 assay

Cells were seeded into 96-well plates (1,500 cells/well). To assess cell proliferation, cells were subjected to Cell Counting Kit-8 (CCK-8) assays (Dojindo, Kumamoto, Japan) at 0, 12, 24, 36, 48, 60 and 72 h. Each time, supernatant was replaced by RPMI-1640 medium containing 10% CCK-8 reagent. After 2 h incubation at 37°C, the absorbance at 450 nm was measured to determine the number of viable cells, according to the manufacturer's protocol. The experiment was repeated three times independently. The cell doubling time (DT) was calculated by the equation: TD=ΔTx[lg2/(lgNt-lgN0)], where ΔT is the time interval; N0 is the initial cell number and Nt is the end point cell number.

Mouse primary OBs (4 ​× ​10³ ​cells/per well) were seeded in 96-well plates. 200 ​μL α-MEM containing SGPA (0, 1, 5, 10, 20, 50, 100, 200 ​μM) were added to each well after the cells adhered to the wells. Cell viability was tested after culturing for 48 ​h. The plates were rinsed with phosphate buffered saline (PBS, HyClone, USA) before the Cell Counting Kit-8 (CCK-8) assays (Dojindo, Japan). According to the manufacturer's instructions, 10 ​μL CCK-8 reagent and 90 ​μL α-MEM were added into each well, further incubated at 37 ​°C for 2 ​h. The OD of cells at 450 ​nm was measured by a microplate reader (Varioskan LUX, Thermo Fisher Scientific, MA, USA). Cell viability was calculated as (OD _SGPA-OD _blk)/(OD _ctrl-OD _blk) ​× ​100% (blk ​= ​blank, ctrl ​= ​control).

SCC12 cells (5×10⁴ cells/well) were seeded into 24-well plates and incubated at 37°C for 24 h, after which the cells were treated with increasing concentrations of GSP (10, 50, 100 and 200 µg/ml) for 24 h. The cell viability was assessed using Cell Counting kit-8 (CCK-8) assays (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, CCK-8 solution (10 µl) was added to each well and incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO₂. Absorbance was then measured at 450 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The effect of 3-MA on cell death was determined after the cells had been treated for 24 h. Cells were pre-incubated with 3-MA (10 mM) for 1 h prior to the addition of GSP.

Wound healing assays were performed to detect the migration ability of HNSCC cells. Transwell assays including uncoated inserts and Matrigel‐coated inserts were used separately to determine migration and invasion abilities. The proliferation ability was determined using Cell Counting Kit 8 (CCK8) assays (Dojindo, Japan). Finally, colony‐forming abilities were compared by the number of clusters formed by 1000 cells incubated for 10–14 days. These experiments were performed as described in our previous study and were all repeated three times (Xu et al., 2020 (link)).

A progesterone-resistant Ishikawa cell line (Ishikawa-PR cells) was obtained from parental Ishikawa cells via continuous exposure to increasing amounts of MPA dissolved in DMSO18 (link). Under these conditions, the proliferation of cells was the same as parental Ishikawa cells, indicating an acquisition of resistance to the growth inhibitory effect of MPA. Monoclonal-resistant cell lines were obtained from a pool of resistant cells by serial dilutions. The Cell Counting Kit-8 (CCK-8) assays (Dojindo, Kumamoto, Japan) were used to verify the establishment of the Ishikawa-PR cell line. Parental and Ishikawa-PR cells were seeded in a 96-well plate at a density of 3,000 cells/well and cultured for 1–7 days. CCK-8 assays were added to each well every 24 h, and the cells were cultured for an additional 1 h. The optical density (OD) value for each well was read at 450 nm using an automated microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were repeated three times.