Cell counting kit 8 cck 8 kit

For these assays, five thousand cells were seeded in 96-well flat plates. After twelve hours of cell seeding to allow cell attachment to the plates, each cell line was treated with curcumin, andrographis, or their combination using different concentrations for forty-eight hours. After treatment, the cell viability assays were performed using the Cell Counting Kit-8 (CCK-8) kits (Dojindo, Kumamoto, Japan). A quantity of 10 μL of the CCK-8 reagent was added to each well, and the cells were incubated in a 37 °C incubator for two hours. After that, the absorbance of the cells was analyzed at 450 nm using an enzymatic plate analyzer (Molecular Devices, San Jose, CA, USA).

Newborn Calf Serum (NCS), phosphate-buffered saline (PBS) (BI, Beit Haemek, Israel), Trypsin-EDTA Solution (Solarbio, Beijing, China). RPMI 1640 cell culture medium (Corning, Steuben County, New York, U.S.A.), Hochest, Mitotracker dye (Thermofisher Science, MA, U.S.A.). Experimental dimethyl sulfoxide (DMSO) is derived from the Sigma (# d2650, U.S.A.). Cell Counting Kit-8 (CCK-8 kit ) (Dojindo, Japan).

The proliferation ability was analysed using the Cell Counting Kit-8 (CCK-8 Kit, Dojindo Laboratories, Japan) assay. Glioma cells (5000 cells/well) were seeded into 96-well plates (Corning, USA) at 100 μl and incubated overnight. Then, the medium was changed to serum-free DMEM medium, and TMZ was added as scheduled. Ten microlitres of CCK-8 solution was added at various time points, and the absorbance value at 450-nm wavelength was detected by a microplate reader (PerkinElmer, USA) after 2 h of incubation. The migration ability was investigated using a wound-healing assay. Glioma cells were seeded into 6-well plates and incubated until they reached 90–100% confluence. A 10-μl pipette tip was used to carefully make cross lines, and the debris was washed away with PBS. Different medium with 200 μM TMZ was added. The areas of the scratch wounds were imaged with an Olympus microscope at 0 and 24 h and analysed using ImageJ software (NIH, USA). The apoptosis ability was analysed by flow cytometry (FCM). Glioma cells were treated with TMZ for various times and then collected. Glioma cells were washed and processed to FCM by using an annexin V-APC/7-AAD or annexin V-FITC/PI apoptosis assay kit (Nanjing KeyGEN, China) in accordance with the manufacturer’s instructions.

HK2 cells were planted at a density of 1.0χ10³/well in 96-well plates. Before detection, the culture medium was evacuated, and cells were stained with Cell Counting Kit-8 (CCK-8 kit, #CK04, Dojindo, Kumamoto, Japan). Thermo Scientific Multiskan FC (ThermoFisher, Waltham, MA, USA) was used to measure the absorbance at 450 nm after 1 hour of incubation at 37°C (Waltham, MA, USA). The following formula was used to calculate cell proliferation activity: proliferation activity (%) = [A (delivery) – A (blank)]/[A (control) – A (blank)] multiplied by 100.

Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Inc., China) according to the manufacturer’s protocol. For the cell migration assay, A549 cells were suspended in 1% FBS DMEM, incubated at 37 °C for 2 h, and then placed in the upper transwell chambers. The lower chambers were filled with DMEM with 5% FBS. After 24 h, the inserts were removed, and inner side was wiped with a cotton swab. The cells were fixed with 70% ethanol, stained with Giemsa, and counted under light microscopy.