Cell counting kit 8 assay

The viability of SZ95 was assessed using Cell Counting Kit (CCK)-8 assays (Dojindo, Kumamoto, Japan). The SZ95 cells were seeded in 96-well culture plates at 5.0 × 10⁴ cells·mL⁻¹ and allowed to attach for 24 h. The medium was replaced with serum-free media. Cells were treated with serum-free media containing different concentrations of BV (1, 10 and 100 ng·mL⁻¹) and melittin (0.1, 0.5 and 1 μg·mL⁻¹) for 8 and 16 h. After experimental treatment, 10 μL of the WST-8 solution (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) was added to each well, and the SZ95 cells were incubated for an additional 2–4 h at 37 °C. The absorbance values were measured at 450 nm using a microplate reader.

The cell proliferation and viability of RAW264.7 cells were evaluated by Cell Counting Kit (CCK)-8 assays (Dojindo Molecular Technologies, Tokyo, Japan). RAW264.7 cells (3 × 10³ cells/well) were seeded onto 96-well plates at 2 × 10³cells/well for 24 h and 72 h after treating with different concentrations of IFN-λ1. A 10% CCK-8 solution was added into each well, and the plates were incubated at 37 °C in a 5% CO₂ atmosphere for 2 h. The absorbance at 450 nm was detected on the instrument (BioTek, Synergy H4, USA).
For detecting the effect of IFN-λ1 on cell apoptosis, we have used flow cytometry (BD, Triangle, NC, USA). The RAW264.7 cells were seeded onto 6-wells plate, they were incubated and divided into 50 ng/ml M-CSF group, 100 ng/ml RANKL + 50 ng/ml M-CSF group, 100 ng/ml RANKL + 50 ng/ml M-CSF group + 100 ng/ml IFN-λ1 group, and 100 ng/ml RANKL + 50 ng/ml M-CSF group + 200 ng/ml IFN-λ1 group. These groups were incubated for 48 h. The Annexin V-FITC/PI staining was performed to detect apoptosis of cultured cells. The process is consistent with the instructions (Life Technologies, USA).

Cell proliferation was assessed by Cell Counting Kit-8 assays (CCK-8; Dojindo Molecular Technologies, Japan). The transfected cells (Caco-2 and LOVO) were cultured in 96-well plates (5 × 10³cells/well). Then, each well was incubated with CCK-8 reagent at 0, 24, 48, 72, and 96 h, and cells were further cultured for 2 h at 37 °C. Absorbance at 450 nm was read using a microplate reader. All tests were repeated three times.

The BMSCs were plated in 96-well plates (5 × 10⁴ cells/ml, 100 μl per well) and grown overnight. The cells were treated with 200 µM H₂O₂ and different concentrations of tocopherol (Sigma, United States) (from 1 to 200 µM) for 4 h. Then, each well was cultured with a normal medium for 24 h. The cell viability was stained with Cell Counting Kit-8 assays (CCK-8, Dojindo, Japan), and the OD value was determined at 450 nm by using a microplate reader (Bio-Rad, United States).

The cell proliferation rate was detected using Cell Counting Kit-8 assays (CCK-8, Dojindo) according to the instructions. 100 ul cell suspension (1×10³ cells/well) was seeded in a 96-well plate and various concentrations of substances were added. The cells were incubated for an appropriate length of time (24, 48, and 72 h) and obtained with 10ul CCK-8 solution added. Absorbance was measured using a microtiter plate reader at 450 nm.

In vitro cell viability was detected using Cell Counting Kit-8 assays (Dojindo, Japan). Transwell assays were performed at 48 h after transfection: 1 × 10⁴ cells were resuspended in serum-free Opti-MEM medium, and cell invasion/migration was examined in Transwell cell culture chamber filters coated on the upper side with/without Matrigel (Corning Biocoat) as previously described [34 (link)]. The wound healing assays we performed by using culture insert (ibidi, Germany, 80,206) according to the instructions of the manufacturer. The apoptotic rates of SiHa and CaSki cells were determined at 72 h after transfection using an Annexin V-FITC/PI Apoptosis Kit (Mutisiences, China, AP101–100-kit). For cells transfected with circCDKN2B-AS1-overexpressing or empty plasmids, apoptosis was induced by incubation with serum-free medium for 6 h.