Cell counting kit 8 cck 8 assay

The cytotoxicity of L. mesenteroides LVBH107 for RAW 264.7 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay (APExBIO, Houston, TX, USA). After RAW 264.7 cells were inoculated into wells of 96-well plates and allowed to adherently grow on well surfaces to 70–80% confluence (1 × 10⁶ cells/well), different concentrations of live or heat-killed L. mesenteroides LVBH107 were added to the wells (0, 7, 8, and 9 Log CFU/mL) followed by co-incubation of plates for 6 h at 37 °C. Thereafter, the culture medium was removed from the wells; then, 300 μL of CCK-8 working reagent was added to each well, followed by the incubation of plates at 37 °C for 4 h. Next, the optical density of each well at 450 nm was measured using a multi-function microplate reader (F200 Pro, Tecan Infinite, Männedorf, Switzerland). Cell viability was calculated as follows:
where As is the OD₄₅₀ value of RAW 264.7 cells co-incubated with L. mesenteroides LVBH107, and Ac is the OD₄₅₀ value of RAW 264.7 cells in the absence of L. mesenteroides LVBH107.

The Cell Counting Kit-8 (CCK-8) assay (APExBIO, Houston, TX, USA) or the MTT assay kit (Amresco, Boise, ID, USA) could detect cell viability based on the manufacturer’s instructions using 96-well plates with 2000 cells per well.

A cell proliferation assay was performed with the Cell Counting Kit-8 (CCK8) assay (APExBIO). Equal numbers of HCT116 and SW620 cells 24 h after transfection with si-MK5-AS1, si-MK5, let-7f-1-3p inhibitor and let-7f-1-3p mimics were seeded into 96-well plates. CCK8 solution (10 μl per well) with 100 μl serum-free medium was added every 24 h, and the plates were incubated at 37 °C for 2 h. The optical density (OD) was then measured at 450 nm. For the colony formation assay, HCT116 and SW620 cells transfected similarly were plated in each well of a six-well plate and cultured in the appropriate medium containing 10% FBS for approximately 14 days, and the medium was replaced every 5 days. After 14 days, the colonies were fixed with methanol and stained with 0.1% crystal violet (Vicmed, China). The colony formation rate was determined by counting the number of stained colonies.

Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay (APExBIO, USA) and 5-ethynyl-20-deoxyuridine (EdU) incorporation assay (Beyotime, China). Briefly, PC12 cells were plated in 96-well plates at a density of 5 × 10³ cells per well with 100 μl of complete culture medium. After 24 h, cells were treated with the indicated amount of ADSC-exo, as previously described. After continuous culture for 2 days, 10 μl CCK-8 solution was added to each well and incubated for 1 h at 37°C. The absorbance value at 450 nm was detected by a microplate reader (Thermo MK3, USA).
For the EdU incorporation assay, PC12 cells were seeded in 6-well plates at a rate of 5 × 10⁵ cells/well. After 24 h, cells were treated with the indicated amount of ADSC-exo, as previously described. After continuous culture for 24 h, cells were treated with EdU solution (Beyotime, China) for 2 h and incubated in click reaction solution for 30 min in the dark at 37°C. Cells were stained according to the manufacturer's instructions, then imaged under a fluorescence microscope, and the percentage of EDU-positive cells was calculated.

After treatment with different concentrations of CXCL10 for 24 h, the cell viability of hGL cells was quantified by the Cell Counting Kit-8 (CCK-8) assay (ApexBio, China). The absorbance at 450 nm was read on the microplate reader.