Cell counting kit 8 (cck8)

Cell Counting Kit-8 (CCK-8, KeyGen) was applied to evaluate cell proliferation. Experiments were performed according to the manufacturer’s protocol. Briefly, 1 × 10⁴ cells were seeded in a 96-well plate containing 100 μL of completed culture medium per well and incubated in a 37 °C incubator. Culture medium was used as a blank control. Cell proliferation was evaluated every day for approximately 1 week after plating. CCK-8 solution (10 μL) was added to each well, and then, the plate was incubated with cells in the 37 °C incubator for 2 h. An optimal density (OD) value of 450 nm was used to measure cell proliferation. The mean and SD were calculated from 3 independent assays.

The cell proliferation was monitored using a Cell Counting Kit-8 (KeyGEN, Nanjing, China) or the xCELLigence system. After transfection, cells were plated in 96-well plates at a density of 2000 cells in 100ul per well and the absorbance was measured at 450 nm with an ELx-800 Universal Microplate Reader. Experiments were repeated at least three times with similar data. For the xCELLigence system, exponentially growing cells with corresponding treatment in complete media were seeded in E-plates at a density of 20,000 per well. The plates were then locked into the RTCA DP device in the incubator. The proliferative ability in each well was automatically monitored by the xCELLigence system and expressed as a “cell index” value. The cell growth was recorded in real-time for 90 h.
For colony formation assay, a total of 100 transfected cells were placed in a fresh 6-well plate and maintained in media containing 10% FBS, replacing medium every 3 or 4 days. After 2 weeks, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Visible colonies were then counted. For each treatment group, each well was assessed in triplicate.

Cell proliferation was evaluated using Cell Counting Kit-8 (Keygen, China) following the manufacturer's instructions. ASC, HUVEC and RAW264.7 were seeded onto 96-well plates with the density of 2.5 × 10³, 500 and 10⁴ cells per well, respectively. Then the cells were treated with 50 μg/mL sEV-AT or ATE (0.1 mL per well). Growth curves were plotted according to the OD value which was measured at 450 nm using a spectrophotometer from day 1 to day 6 (n = 5).

Cell proliferation under different stimulation conditions was assessed using a cell counting kit-8 (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) according to the manufacturer’s protocol.

RKO and Ht29 cell lines were purchased from the American Type Culture Collection. DSF was procured from Macklin. In previous studies, it is demonstrated that the anticancer function of DSF is dependent on the presence of a Cu²⁺ ion (Skrott et al., 2017 (link); Huang et al., 2021 (link)). In our study, we establish that 10 μM Cu (Copper (II) chloride dihydrate, Solarbio) is nontoxic in vitro and in vivo. In vitro cytotoxicity analysis was performed according to manufacturer’s instructions: RKO and Ht29 cells (7000 cells/well) were cultured overnight in 96-well plates; exposed to DSF (0.25 μM), Cu (10 μM), or a combination of both for 24, 48, and 96 h; and then analyzed for cell viability using the Cell Counting Kit-8 (KeyGEN BioTech, Nanjing, China). In parallel, CRC cells were planted into a six-well plate containing 5 ml medium at a density of 500 cells/well and exposed to DSF, Cu, or a combination of both for 96 h. Then, the cells were rinsed with 0.01% PBS, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet. The number of clones was counted using Image J software.

The 293T and HepG2 wild‐type and knockout cell lines were trypsinized, seeded into 96‐well plates at a concentration of 1500 cells per well in triplicate, and cultured at 37°C. Cell counting kit‐8 (KeyGen, China) was added after 18 hours and incubated for 1 hour. The absorbance was measured at 450 nm every other day until one of the cells reached the confluence of more than 70% to stop the cell proliferation experiment.