Tween 20

The differentiated L6 cells were in serum-removed medium for 2 h. L6 cells were slowly washed with pre-cooled PBS and solubilized with RIPA lysate (Cat# P0013B, Beyotime, Shanghai, China) containing PMSF protease inhibitor cocktail (Cat# ST506, Beyotime, Shanghai, China) and phosphatase inhibitor cocktail (Cat# B15001, bimake, Shanghai, China) for 30 min on ice. The mixture was centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatant absorbed was total cellular protein. The lysate was added relative to SDS-PAGE (Cat# P0015L, Beyotime, Shanghai, China) protein loaded buffer, and denatured on a metal heater at 95 °C for 10 min. These separated proteins were electrophoretically transferred to a nitrocellulose filter (Pall, Shanghai, China) membrane. Then the nitrocellulose filter membrane was blocked with 5% skim milk (Cat# GC310001, Servicebio, Wuhan, China) in TBS with Tween-20 (BioFroxx, Guangzhou, China) solution for 2 h. Lastly, the NC membranes were incubated with the first antibodies overnight and the second antibodies for 1 h. The ChemiDoc XRS (Bio-Rad, CA, USA) was used to image, and the gray value of protein was calculated by Image J software.

The 293T cells were seeded in cell culture dishes containing complete culture medium Dulbecco’s Modified Eagle Medium (DMEM, Gibco, United States) with 10% fetal bovine serum (FBS, Gibco, United States) and placed in a humidified environment at 37°C with 5% CO₂. After 2 days of cultivation, the cells were digested using 0.25% trypsin (Gibco, United States) and subsequently suspended in Phosphate Buffer Saline (PBS, Gibco, United States) containing penicillin-streptomycin (Gibco, United States). Finally, a cell solution with a concentration of approximately 10⁶ mL⁻¹ was prepared and set aside. This cell solution was injected into the microchannels at a rate of 5 μL min⁻¹ using a syringe pump (Pump 11 Elite, Harvard Apparatus, United States). The microfluidic flow was observed by utilizing polystyrene particles (YUAN BIOTECH, China) with a diameter of 2 μm. To prevent polystyrene particles from adhering to the bottom of the microchannels, the experiment employed 0.2% w/w Tween 20 (Biofroxx, Germany) as a surfactant.

RIPA lysis buffer with 1 mM protein phosphatase inhibitor was added into rCHs for protein extraction. The lysate was collected and centrifuged for 10 min at 12,000 r/min at 4°C. A BCA protein assay kit (Thermo Scientific) was used to determine the concentration of protein. The proteins were separated by 10% SDS-polyacrylamide gels, and then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The block solution was the TBS (Tris-HCL Buffer Solution) containing bovine serum albumin (0.5%) (BSA, Sigma-Aldrich, Darmstadt, Germany) and Tween-20 (0.1%) (Bio Froxx, Guangzhou, China). The membranes were blocked with block solution for 1 h at room temperature, incubated with antibodies against matrix metalloproteinase 13 (MMP13, 1:1000, GTX100665, GeneTex, CA, USA), CollagenII (1:1000, ab34712, Abcam, Cambridge, UK), cyclooxygenase-2 (COX2, 1:1000, #12282, Cell Signaling Technology, Danvers, USA), TLR2 (1:1000, #29071, SAB, Nanjing, China), p-NF-κB (1:1000, #3033T, Cell Signaling Technology, Danvers, USA), NF-κB (1:1000, #8242T, Cell Signaling Technology, Danvers, USA) at 4°C overnight. Next, the TBST was used to wash membranes and the membranes incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The ChemiDocXRS + Imaging System (Tanon, Shanghai, China) was used to detect the signals. The protein bands were quantitatively analyzed by ImageJ.