Cck 8 kit

Approximately 2 × 10⁵ 2BS cells were seeded into 24-well plates and
incubated overnight in a CO₂ incubator. Inhibitors were serially
diluted concentrations (0, 0.008, 0.032, 0.125, 0.5, 2, and 8 μM) and then
transferred to the plate containing 2BS cells. Seven days after addition of
drug, CCK-8 kit (Sangon Biotech) was used according to the manufacturer’s
protocol. In brief, each well of the plate had10 μl CCK-8 solution added and was
incubated 2 h at 37°C. Absorbance at 450 nm was measured by SynergyH1 microplate
reader (BioTek).

The agonist PNU282987 and antagonist MLA were purchased from Tocris Bioscience (Bristol, UK), and LPS was purchased from Sigma–Aldrich (St. Louis, MO, USA). The [A10L]PnIA linear crude peptide was synthesized by Bankpeptide Biological Technology Co., Ltd. (Hefei, China) and purified in our laboratory.
A CCK-8 kit, DMEM-H medium, Penicillin/Streptomycin Solution, and PBS buffer were purchased from Sangon Biotech (Shanghai, China). FBS was purchased from Procell (Wuhan, China). A FastPure Cell/Tissue Total RNA Isolation Kit, HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper), 2 × Taq Plus Master Mix Ⅱ, and ChamQ Universal SYBR qPCR Master Mix were obtained from Vazyme (Nanjing, China). A TNF-α ELISA kit, IL-6 ELISA kit, and IL-1β ELISA kit were purchased from Thermo Scientific (Waltham, MA, USA).

Cell toxicity and proliferation were measured using the CCK-8 kit (Sangon Biotech Co. Ltd., Shanghai, China). To determine toxicity, NP cells seeded in 96-well plates at a density of 8 × 10³ cells per well were cultured for 24 h with DHE at concentrations of 0, 2.5, 5, 10, 20, or 40 μM. To determine proliferation, NP cells seeded in 96-well plates at a density of 2 × 10³ cells per well were cultured for 8, 24, 48, and 72 h. At 8 h, optical density values were recorded on a spectrophotometer, with 450 nm set as the baseline. Then, DHE concentrations of 0, 2.5, 5, 10, 20, or 40 μM were added into the plates. At the end of each experimental period, cells were incubated with 10 μL of the CCK-8 reagent for 1 h at 37°C. Optical density values were recorded on a spectrophotometer at 450 nm on an Infinite M200 Pro multimode microplate reader (Tecan Life Sciences, Männedorf, Switzerland).

The effect of the HOXA-AS2/miR-885-5p/RBBP4 axis on cell viability was assessed using the CCK-8 assay. Transfected U87 and U251 cells (100 μL) in the logarithmic growth phase were seeded into the 96-well plates at a density of 2000 cells/well before being incubated at 37 °C. After an incubation period of 0, 24, 48 and 72 h, 10 μL CCK-8 solution was added to each well. The cells were later incubated for 2 h according to the instruction manual of the CCK-8 Kit (Cat#: E606335, Sangon, China). The absorbance was measured at 450 nm with a microplate reader to assess cell viability.

The recombinant plasmid pPIC9K::FIP-glu-His and Pichia pastoris GS115 strain were preserved in our laboratory. HisSep nickel-nitrilo-triacetic acid (Ni-NTA) agarose resin was purchased from Yeasen (Shanghai, China). The restriction enzyme Sac I was purchased from Takara (Beijing, China). DPPH and vitamin C were purchased from Innochem (Beijing, China). The CCK-8 kit and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and the Bradford protein assay kit were procured from Sangon (Shanghai, China). Other reagents were of analytical grade.

NP cells' toxicity and proliferation were detected using the CCK-8 kit (Sangon Biotech Co., Ltd., Shanghai, China). To determine toxicity, NP cells seeded in 96-well plates at a density of 2 × 10⁴ cells per well were cultured for 24 h with eupatilin at concentrations of 0, 1.56, 3.12, 6.25, 12.5, 25, 50, or 100 μM. To determine proliferation, cells seeded in 96-well plates at a density of 2 × 10³ cells per well were cultured for 24, 48, 72, and 96 h with eupatilin at concentrations of 0, 1.56, 3.12, 6.25, 12.5, 25, 50, or 100 μM. At the end of each experimental period, the medium was replaced with 10 µl of CCK-8 reagent and 90 µl of complete DMEM for 2 h in a 37°C incubator. Then, the optical density values of each well were recorded on a spectrophotometer at 450 nm on an Infinite M200 Pro Multimode Microplate Reader (Tecan Life Sciences, Männedorf, Switzerland).