Cell counting kit 8 cck 8 assay kit

Cell vitality was detected using the Cell Counting Kit-8 (CCK-8) assay kit (Bimake). Briefly, transfected cells were seeded into 96-well plates and incubated with CCK-8 reagent every 24 hours according to the manufacturer’s instructions. For colony formation assay, cells after transfection were cultured in 6-well plates, and after 14 days, the cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were photographed. Cell proliferation was identified using Ethynyldeoxyuridine (EdU) assay following the manufacturer’s protocol of 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit (Ribobio, Guangzhou, China). The transfected cells were treated with 50 µM Edu labeling medium and incubated for 2 hours. Next, the cells were fixed with 4% paraformaldehyde and the cell membrane was permeated with 0.5% Triton X-100. Subsequently, cells were added with anti-Edu working solution and DAPI staining solution. Edu positive cells were identified and counted under fluorescent microscopy. The percentage of Edu positive cells was calculated from five random fields in three wells.

Cell growth was assessed by the Cell Counting Kit‐8(CCK‐8) Assay Kit (Bimake). The experiments were repeated six times. For the colony formation assay, 500–1000 cells were seeded into each well of a six‐well plate with soft agar (Agarose; Sigma) and maintained in a medium containing 10% FBS for 10–14 days. The colonies were stained with 0.1% crystal violet. The number of clones containing at least 20 cells was counted using an inverted microscope.

Primary HRVECs were obtained from ScienceCell (Carlsbad, CA) and cultured with ECM medium (Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Shanghai, China) containing growth factors and 5% FBS in a humidified incubator at 37°C with 5% CO₂. Cell Counting Kit-8 (CCK-8) assay kit (Bimake, Shanghai, China) was used to measure the effects of HCQ on the cell viability of RVECs: RVECs were seeded in 96-well plates with HCQ (0, 20, 40, 80 and 100 µM) for 48 h. At that time, cells were added with CCK-8 reagent diluted at a 10:1 ratio and cultured for another 2 hours before the OD value of each well was measured at 450 nm using a Synergy H1 microplate reader (BioTek, Vermont, USA). Cells were pretreated with HCQ at nontoxic concentrations for 10 h at 80%–90% confluence, and then incubated with recombinant TNF-α (10 ng/mL, Life Technologies, Carlsbad, CA) for different time according to experimental requirements.