Cck 8

Raw264.7 and Caco-2 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), which were grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Solarbio, Beijing, China), containing 10% (v/v) fetal bovine serum (FBS) (Biological Industries, Israel) and 1% (v/v) penicillin/streptomycin (Solarbio, Beijing, China) under an atmosphere of 5% CO₂ at 37 °C. RAW264.7 and Caco-2 cells were incubated in 96-well plates (2 × 10⁴ cells/well), respectively, and L. plantarum strains dispersed in DMEM without FBS and antibiotics at a concentration of 1 × 10⁷ CFU/mL were added to plates. After intervention for 24 h, 10 µL of CCK-8 (GlpBio, Shanghai, China) was added into wells and incubated at 37 °C for 2 h, and the absorbance was measured at 450 nm [24 (link)].

CCK-8, an assay kit produced by GLPBIO (United States), was used to perform cell viability examinations based on the manufacturer’s guidelines. Cells were first treated with the miR-21-5p suppressor and/or reversine. They were then seeded into 96-well plates at a density of 3 × 10³ cells/well. After incubating the cells for different durations (24, 48, and 72 h) in an atmosphere containing 5% CO₂ at 37°C, the CCK-8 reagent was added to the 96-well plate and incubated for another 2 h. A microplate reader (Bio-Rad, USA) was used to read the absorbance of the sample at 450 nm.

To analyze the effects of DHFR, MSH3, ZFYVE16 and FAM151B on MTX resistance, cells transfected with target gene siRNAs and control siRNA were harvested and seeded into 96-well plates at a density of 3000 cells per well. The cells were then treated with MTX for 48 h. Following this incubation period, the reagent of CCK-8 (GLPBIO, Montclair, CA, USA) was added to each well, and the plates were incubated for 2 h. Absorbance at 450 nm was measured to calculate the IC₅₀ value for MTX.

We used cell counting kit-8 (CCK-8, Glpbio, CA, USA). assay and flow cytometry analysis to assess the potential cellular cytotoxicity of PDA NPs. HaCaT cells were seeded and cultured overnight, then incubated with different concentrations of PDA NPs for 24 h. In the CCK-8 assay, the cells were rinsed three times with PBS. Subsequently, 100 μL CCK-8 working solution (CCK-8: MEM = 1:10) was added to each well and incubated for 2 h. The optical density (OD) was measured at 450 nm using a microplate reader (Multiskan GO, ThermoScientific, America) and the cell viability was calculated as (OD test group – OD blank group) / (OD control group – blank group) × 100%. For flow cytometry analysis, HaCaT cells were stained with Fixable Viability Dye eFluor^™ 780 (eBioscience) according to the manufacturer’s instructions.

NPCs were seeded in 96-well plates at a density of 5,000 cells per well for 24 h. Then, NPCs are treated with different concentrations of DHA as shown in Figure 1(a) and cultured for 0, 24, 48, 72, and 96 h. At the indicated time, 100 μL fresh medium with 10 μL CCK-8 (Glpbio) was added to each well. After incubated for 2 h, the absorbance of 96-well plates was measured by a microplate reader (Tecan Sunrise, Salzburg, Austria) at 450 nm. The cell viability was calculated according to the manufacturer's instruction.