Cck 8 assay kit

Cell counting kit-8 (CCK-8) Assay Kit was bought from GlpBio (Montclair, CA, USA). After treating with ox-LDL or transfecting with plasmids and oligonucleotides for 24 h, HUVECs were digested with trypsin (Solarbio) and inoculated into 96-well plates. After 24 h, CCK8 solution was added into cells and further cultured for 4 h. Finally, the absorbance at the wavelength of 450 nm was detected by a microplate reader, and the cell viability was calculated.

Cellular viability was detected with a CCK-8 assay kit (GLPBIO, California, United States). The HSC-T6 cell line was planted into the 96-well culture plate, followed by 24 h of exposure to various doses of For, Iso, Cal, Que and Kae (0.1–200 μM). Thereafter, 10 μl CCK-8 solution was placed into all wells to incubate for 1 h, and the microplate reader (Thermo Varioskan LUX, MA, United States) was used to measure the absorbance (OD) value at 450 nm.

CCK-8 assay applied to the detection of cell viability, and CCK-8 assay kit (GLPBIO, GK10001, California, America) was used to detect cell growth at 0 h, 24 h, 48 h, and 72 h. The initial number of cells per well is 1000. The absorption value at 450 nm was tested, and then the growth curve was drawn according to the absorption value.

Cellular viability was evaluated using the CCK-8 assay kit (GLPBIO, California, United States). The HSC-T6 cells were cultured in 96-well culture plates and then treated with different concentrations of AHWE ranging from 0 to 2.5 mg/ml for 24 h. Next, the CCK-8 solution (10 µL) was added to each well, and the absorbance was measured after 1 h of incubation using a microplate reader (Thermo Varioskan LUX, MA, United States) at 450 nm. Each experiment was conducted in triplicate for each group. Percent viability was calculated as the absorbance of the cells treated with different doses of AHWE relative to that of the control cells.

Chitosan with low molecular weight (50–190 kDa based on viscosity, 75–85% deacetylated) and sodium tripolyphosphate were purchased from Sigma–Aldrich (St. Louis, MO, USA). d(+)‐trehalose dehydrate (99%) and FITC, isomer 1 (95%), were purchased from Alfa Aesar (Tewksbury, MA, USA). Trehalose assay kit was purchased from Megazyme (Wicklow, Ireland). Eagle's minimum essential medium (α‐MEM), Roswell Park Memorial Institute (RPMI)‐1640 medium, horse bovine serum, 2‐mercaptoethanol (2‐mer, 50 × 10⁻³m), and penicillin and streptomycin were purchased from Gibco (ThermoFisher Scientific, USA). Myo‐inositol and folic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). FBS and DMSO were purchased from Corning (New York, NY, USA). Recombinant human IL‐2 (rhIL‐2) was gifted from Akron Biotech (Boca Raton, FL, USA). The CCK‐8 assay kit was purchased from Glpbio (Montclair, CA, USA). The CFSE/7‐AAD kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). All the antibodies including PerCP‐Cy5.5‐conjugated IFN‐γ antibody (clone B27), phycoerythrin (PE)‐conjugated‐CD107a antibody (clone H4A3), GolgiStop solution, Cytofix/Cytoperm solution, and Perm/Wash buffer were purchased from BD Biosciences (San Jose, CA, USA). All other chemicals were purchased from Sigma, unless specifically noted otherwise.

For CCK-8 analysis, cells were seeded at a density of 3 × 10³ cells per well in a 96-well plate and incubated overnight, following the instructions provided in the CCK-8 assay kit (GLPBIO, Montclair, CA, USA). On the following day, chrysin was added to the wells and, after a treatment duration of 24, 48, or 72 h, assessments were conducted. For the cell counting assay, cells were seeded at a density of 3 × 10⁵ cells per well in a 6-well plate and incubated overnight. On the next day, corresponding treatments were carried out as per instructions, followed by an additional 48 h incubation before cell counting. For clonogenic assays, cells were seeded at a density of 1 × 10³ cells per well in a 6-well plate and incubated overnight. The following day, after cell adhesion, drug treatment was administered for 4 h. After 4 h, the medium was replaced with fresh culture medium, and cells were cultured for two weeks. Once distinct colony formation was observed, fixation was performed using 4% paraformaldehyde (NCM Biotech, Suzhou, China), followed by staining with 0.5% crystal violet (Beyotime, Shanghai, China) overnight, and finally imaging and quantification.