Microplate autoreader

The minimal inhibitory concentration (MIC) of the biogenic MccJ25 was determined in sterilized 96-well microplates (Costar, Corning Inc., Corning, NY, USA) using microdilution assays as described previously (Wang et al., 2017 (link)). Briefly, biogenic MccJ25 was dismissed in distilled water, and serial 2-fold dilutions were made in MH broth using 96-well microplates. Finally, the concentration of biogenic MccJ25 ranged from 0.125 to 256 μg/mL. Ten mL of bacteria stay overnight culture mixture was inoculated into each well at a concentration of 1.0 × 10⁶ CFU/mL. The micro plate was hatched at 37°C for 24 h and bacterial growth was surveyed by an alter in absorbance at 600 nm using a microplate auto reader (Bio-Rad Laboratories, Hercules, CA). Positive (media with inoculum) and passive controls (media only) were included. The MIC was determined as the lowest concentration of biogenic MccJ25 that restrained bacteria growth (be short of raise in absorbance reading). The dissect were performed in triplicate.

Sh-control and sh-PLK4 NB cells were seeded in 96-well plates at a density of 3 × 10³ cells per well for initial concentration. Each group of cells set five parallel holes. Then, the cells were incubated with 20 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide, 5 mg/mL in PBS, Sigma, St. Louis, USA) were added to each well at 37 °C, 5% CO₂ for 4 h, following which the medium was removed and 150 μL of DMSO (dimethyl sulfoxide, Sigma) was added. The optical density was measured using a micro-plate auto-reader (Bio-Rad). Cell viability was examined at 0, 24, 48, 72, and 96 h.
For colony formation assay, sh-control and sh-PLK4 NB cells were seeded in six-well plates at a density of 0.5 × 10³ cells per well. The cells were allowed to grow at 37 °C, 5% CO₂ for approximately 2 weeks. When they grew to visible colonies, the cells were stained with crystal violet. The colonies were counted and the pictures were taken by digital camera.

Cell counting Kit-8 (CCK-8) assay and 5-Ethynyl-20-Deoxyuridine (EDU) incorporation assay were used to evaluate the chondrocytes proliferation abilities. For the CCK-8 assay, as previously reported [71 (link)], chondrocytes were seeded in 96-well plates (2 × 10³) and supplemented with complete growth medium and followed by different transfection 24 h later. At days 1, 2, 3, 4, and 5 after transfection, 10 μL CCK-8 solution was added into each well and incubated for 2 h. Absorbance was measured at 450 nm with a Microplate Autoreader (Bio-Rad, Hercules, CA, USA). Experiments were repeated in triplicate. For EDU incorporation assay, EDU detection kits (Ribobio, Guangzhou, China) were applied and the detailed processes were performed according to the manufacturer’s instructions.