Ab215188

Total protein was extracted from tissues by RIPA lysis buffer (Beyotime), 10 μL PMSF and 10 μL phosphatase inhibitor. SDS-PAGE gel electrophoresis was utilized to separate the extracted protein. The protein was transferred to polyvinylidene fluoride membrane. After blocking with 50 g/L BAS for 1 h, the membrane was incubated with primary antibodies against HTR2B (1/3000; ab227722; Abcam, Cambridge, MA, United States), SLC5A3 (1/1000; ab113245), GAPDH (1/3000; ab8245), TNF-α (1/1000; ab215188), IL-1β (1/1000; ab200478), TGF-β (9ab208156) and IL-4 (1/1000; ab34277) at 4°C overnight. After washing the membrane with TBST, the membrane was incubated with secondary antibody (1/5000; ab7097) for 1.5 h. The ultra-sensitive ECL chemiluminescence kit was used to configure the luminescence buffer, and the band was developed.

HFLS cells were lysed with RIPA lysate (Beyotime). Total proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with 5% skimmed milk for 1 h, and incubated overnight at 4℃ with primary antibodies against interleukin‐1β (IL‐1β) (1:1000, ab216995; Abcam), interleukin‐6 (IL‐6) (1:1000, ab233706; Abcam), tumour necrosis factor‐α (TNF‐α) (1:1000, ab215188; Abcam), and GAPDH (1:1000, ab8245; Abcam). After washing, the membranes were incubated with a secondary antibody (1:5,000; Biotech) at room temperature for 4 h. The signals were developed using Pierce ECL Western Blot Substrate (Thermo Fisher Scientific), imaged, and analyzed with GAPDH as the control using ImageJ software.

RAW264.7 macrophage cells (ATCC TIB-71) were cultured in DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin. The cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere. For LPS stimulation, RAW264.7 cells were seeded in a 6-well plate at a density of 200,000 cells per well and cultured up to 70–80% confluency. Then, the cells were washed three times with HBSS, followed by 30 min treatment with LPS at a concentration of 100 ng per mL in serum-free medium or with serum-free medium alone as a negative control. Subsequently, the LPS-activated cells were thoroughly washed with HBSS and incubated with 5% and 21% oxygen MSC CGMs for 24 h. The cells without activation were cultured in the same non-conditioned medium.
Next, the cells were washed three times with cold PBS, harvested, and fixed in 0.01% paraformaldehyde for 10–15 min at 4 °C. Afterwards, the cells were permeabilized with 0.2% Tween in PBS for 10 min at room temperature, washed and blocked with 10% FBS in PBS for 20 min, and stained with primary anti-TNFα antibody (ab215188, Abcam, Cambridge, UK) and secondary antibody conjugated with Alexa 647 (ab269823, Abcam, Cambridge, UK). After the staining, the cells were washed three times with PBS to remove unbound secondary antibodies and resuspended in PBS for flow cytometry analysis using CytoFLEX (Beckman Coulter, Brea, CA, USA).