Cck 8

The CCK‐8 (Dojindo) was applied to measure the cell viability. In brief, the cells were cultured at a density of 5000 cells per well in a 96‐well plate. To evaluate the IC₅₀ value of SM on BEAS‐2B cells, the cells were exposed to different concentrations (0, 12.5, 25, 50, 100 and 200 μM) of SM for 30 min. Then, the cells were incubated with new culture medium for 24 h. For the assessment of the effect of HMSCs‐Ex on SM‐exposed cell viability, HMSCs‐Ex with concentrations of 1 × 10⁹ particles resuspended in 150 μL of PBS were added to BEAS‐2B cells after exposure to SM at the concentration of 12.5 μM. After incubation for 24 h, cell viability assay was applied. For the analysis of miRNA inhibitors on cell viability, 10 inhibitors of miRNAs (miR‐100‐5p, miR‐21, miR‐23a‐3p, let‐7a‐5p, miR‐145‐5p, miR‐424‐5p miR‐16‐5p, miR‐24‐3p, miR‐199a‐5p and miR‐15a‐5p) and the control (miR‐NC) were synthesized by Ribo Biotech. The inhibitors were transfected into BEAS‐2B cells using lipofectamine RNAiMAX (Invitrogen) for 48 h. Then, SM was exposed to the cells at the concentration of 12.5 μM and treated with HMSCs‐Ex for 24 h. The CCK‐8 assay was conducted by co‐culturing with 10 μL of CCK‐8 solution and 100 μL of culturing medium at 37°C for 1 h. A microplate reader (BioTek) was applied to quantify the absorbance of the solution by OD values at 450 nm.

The cell viability was detected by CCK-8 (Dojindo Laboratories, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, the GC cells were inoculated to 96-well plates (about 1,000 cells per well) and cultured, and 10 μL of CCK-8 solution was added to each well at 0, 24, 48 and 72 h, and then the cells were incubated with CCK-8 solution for 1 h. Next, the absorbance of the cells was detected at 450 nm with a microplate reader (BioTek, Winooski, VT, USA).

Cell Counting Kit-8 (CCK8; Dojindo Laboratories, Tokyo, Japan) or LDH (Beyotime, Shanghai, China) assay was used to determine cell viability. For CCK8 assay, 24 h after OGD, 10 μl CCK8 was added into each well of 96-well plates, which were incubated at 37 °C for 2 h. Optical density was measured at 450 nm using a plate reader (ELX 800; Bio-Tek, Winooski, VT, USA). For LDH assay, the neurons and culture medium were lysed in PBS containing 1% Triton X-100 at 37 °C for 30 min, respectively. The LDH activities in both the cell lysates and the culture mediums were assayed with the assay kit following the manufacturer’s instructions. LDH leakage was calculated as follows: LDH leakage (%)=LDH culture medium/(LDH culture medium+LDH cell lysates) × 100%.^{73 (link)}

Cell samples were digested by trypsin and made into cell suspension. Appropriate cell density (5 × 10³-2 × 10⁴ cells/well) was inoculated on a 96-well plate for 24-72 h. Cell Counting Kit-8 (CCK-8, DOJINDO, Japan) Cell Proliferation and Cytotoxicity Assay Kit (Roche, USA) were used after cell adherence. CCK-8 detection solutions were added to 96-well plate for 2-4 h, while the absorbance of CCK-8 was read at 450 nm and 630 nm as reference by a full wavelength microarray (BioTek Power Wave XS).

About 1 × 10⁴ transfected PTC cells were seeded in 96-well plates. Cell viability was evaluated with Cell Counting Kit-8 (CCK-8, Dojindo, Japan) at different time points (24, 48, and 72 h) after seeding. After treated with CCK-8 at 37°C for 1 h, PTC cells were used to measure the absorbency at 450 nm using Universal Microplate Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT, U.S.A.).