Cell counting kit 8 cck 8 assay

Cell proliferation was assessed with the cell counting kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology) and 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay (RiboBio Co., China). For the CCK-8 test, cells were plated onto 96-well plates (3 × 10³ cells/well) with omentin-1 (0–800 ng/ml; Omentin Human Recombinant; Prospect, Ness-Ziona, Israel) in a triplicate pattern. Assays were performed from 1 to 7 days after plating with the addition of 100 μl of fresh medium in 10 μl of CCK-8 solution for another 2 h at 37 °C. The optical density (OD) at 450 nm was measured. The assay was repeated three times.
For the EdU assay, cells were incubated with omentin-1 (800 ng/ml) for 5 days in 96-well plates, and then assessed using an EdU assay kit (RiboBio Co., China) according to the manufacturer's instructions. The EdU-positive cells were viewed under fluorescence microscopy (DMI4000B; Leica, Wetzlar, Germany) and the number calculated by counting at least three random separate fields.

For the colony formation assay, cells were plated in 6-well plates. We counted the number of colonies 14 days later and photographed representative wells. In addition, a cell proliferation assay was also performed with the 5-ethynyl-2′-deoxyuridine assay (EDU) Kit (Beyotime, Shanghai, China) and Cell Counting Kit 8 (CCK-8) assay (Beyotime, Shanghai, China) according to the manufacturer’s protocols. The detailed methods were described in our previous research (Hu et al., 2020 (link)).

Cell proliferation rates were tested using a Cell Counting Kit-8 (CCK-8) assay (Beyotime, Jiangsu, China) and a 5-ethynyl-2 deoxyuridine (EdU) assay kit (RiboBio, Guangzhou, China). HUVECs (2000 cells/well) were seeded in a 96-well plate and incubated with different concentrations of hADSC-Exo (50, 100 or 200 μg/mL) or equivalent amount of PBS. Cell proliferation was analyzed at 24 h, 48 h, and 72 h after treatment. A 10 μL CCK8 reagent was added to each well, and the cells were incubated for 2 h at each time point. Then, OD value was verified by a microplate reader (Tecan, Thermo Scientific, USA) at 450 nm. The three independent experiments were performed. Based on the CCK8 data, we determined a suitable hADSC-Exo concentration for the follow-up experiment.
An EdU assay kit was performed to compare the proliferative effects hADSC-Exo and HFF-Exo on HUVEC. HUVECs (5 × 10⁴ cells/well) were seeded in a 96-well plate and cultured to reach 70%–80% confluency. Then, HUVECs were cocultured with the suitable hADSC-Exo concentrations and equal concentration of HFF-Exo for 72 h, and an EdU assay was performed according to the manufacturer's instructions [17 (link)]. Proliferating HUVECs were stained with green fluorescent, and all cell nuclei were stained with blue fluorescent and observed with the Zeiss fluorescence microscope.

After transfection, equal numbers of A549 and Calu‐1 cells were incubated in 96‐well plates (2 × 10³ cells/well) for 24 h. Cell viability was detected by cell counting kit‐8 (CCK‐8) assay (Beyotime) following the manufacturer guidelines as described previously.¹⁵ Optical density at 490 nm was detected using a Benchmark Plus TM microplate spectrometer (BioRad).