C0130

The isolation and use of porcine chondrocytes were approved by the Animal Experimentation Ethics Committee of National Taiwan University Hospital. Fresh porcine stifles were purchased from a traditional market and kept integrated till chondrocytes were isolated under aseptic conditions. A total of 12 porcine stifles were used for all experiments. Porcine chondrocytes were isolated from macroscopically normal cartilage of the femoral condyles [28 (link)]. Finely diced cartilage pieces were treated with 10% antibiotics (15240-062, Gibco, USA) in phosphate buffered saline (PBS) at 37 °C for 10 min, then re-suspended in Dulbecco’s modified eagle’s medium (DMEM; D5648, Sigma, USA) containing 10% fetal bovine serum (12003C, SAFC, USA), 1% penicillin and 0.05% L-Ascorbic acid (A5960, Sigma, USA) and 0.2% collagenase (C0130, Sigma, USA) at 37 °C for 18 h. Chondrocytes were then collected and washed twice with PBS, and cultured in DMEM. Chondrocytes with a passage number of 2 to 4 were used in all experiments.

SVF cells were obtained by collagenase digestion of adipose tissue fragments in Dulbecco’s Modified Eagle’s Medium (DMEM)-Ham’s F12 with Collagenase 11 (Sigma Aldrich C7657, 0.0875 mg/ml), collagenase 1 (Sigma Aldrich C0130, 1.216 mg/ml) and DNAse 1 (Sigma Aldrich DN25, 0.09 mg/ml) under gentle shaking (60 cycles/min) at 37 °C. The resulting suspension was filtered through a 200 μm filter and fat cells were removed as floating fraction after centrifugation (5 min, 1250 rpm). SVF cells were washed once with DMEM-Ham’s F12 to eliminate collagenase, filtered through a 70 μM filter and stained for flow cytometry.

Freshly isolated aortas were resuspended in FACS buffer (PBS containing 1% FBS and 2 mM EDTA) and stained with conjugated antibody for 20 to 30 min at 4 °C. Cells were washed and resuspended in FACS buffer for flow cytometric analyses in which inflammatory cell populations were designated, following gating/stratification of their marker profile. The aortas were cut into small pieces and digested in an enzyme mixture containing 450 U/mL collagenase I (C0130, Sigma‒Aldrich), 125 U/mL collagenase XI (C7657, Sigma‒Aldrich), 60 U DNase I (DN25, Sigma‒Aldrich), and 60 U/mL hyaluronidase (2592, Worthington Laboratories) in PBS with Ca²⁺/Mg²⁺ for 60 min at 37 °C with gentle shaking. After incubation, the digestion mixture was homogenized through a 70-μm nylon mesh. The digestion mixture was centrifuged at 2000 rpm for 15 min at 4 °C, and the cells were simultaneously stained with antibodies at 4 °C for 15 min and then washed and resuspended in a staining buffer. All antibodies are listed in Supplementary Table 3. The cell suspensions were analyzed with a BD FACSCANTO II flow cytometry system (BD Biosciences), and postacquisition analysis was performed with FlowJo7 software (Tree Star).

Female SD rats were euthanatized by CO₂ asphyxia and the freshly excised vaginal tissues were washed and cut into small pieces under antiseptic conditions. The finely minced tissues were subsequently digested enzymatically twice (1 h for each time) with type I collagenase (0.5 mg/mL, C0130, Sigma-Aldrich, USA) dissolved in DMEM/F12 (Gibco, USA) at 37°C, with gentle agitation. The digested tissues were further dissociated and dispersed by repeatedly pipetting and centrifuged at 500 g for 30 s. Afterward, the isolated cell clusters were resuspended and washed twice with DMEM/F12 (Gibco, USA) medium containing 10% FBS (Gibco, USA). The fragments were collected and placed in the six-well plate, then humidified in keratinocyte serum-free medium (K-SFM, Gibco, USA) supplemented with bovine pituitary extract (50 μg/mL, Gibco, USA), recombinant epidermal growth factor (5 ng/mL, Gibco, USA), 100× insulin-transferrin-selenium solution (1×, Gibco, USA), cholera toxin (Sigma Aldrich, USA) and penicillin/streptomycin (100 U/mL/100 μg/mL, Hyclone, USA), in an atmosphere of 5% CO₂ at 37°C. After 24 h, the fragments were washed and cells were cultured with the supplemented K-SFM medium. On day 5, the cells were used for immunofluorescence staining or incubated with live T. vaginalis (1×10⁶) for Western blot assay.

Single myofibres were isolated from Extensor digitorum longus (EDL) muscles as previously described^{34 (link)}. EDL muscles were dissected and incubated in 0.1% w/v collagenase (Sigma, C0130) in DMEM for 1 h in a 37 °C shaking water bath at 40 rpm. Following enzymatic digestion, mechanical dissociation was performed to release individual myofibres that were then fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 5 min at RT, washed three times 10 min with PBS, permeabilised in 0.5% Triton X-100 (Sigma) for 5 min at RT, washed three times 10 min with PBS, blocked in 10% normal donkey serum (DS, Abcam, ab7475) for 1 h at RT, incubated with chicken anti-GFP antibody (1/1000, Abcam, ab13970) and mouse anti-M-Cadherin antibody (1/50, Nanotools, clone 12G4) or goat anti-ItgB1 antibody (1/100, SantaCruz, sc-9936) in 2% DS overnight at 4 °C, washed four times 10 min in PBS at RT, incubated with anti-chicken AlexaFluor 488 (1/500, ThermoFisher, A-11039) and anti-mouse Cy3 antibody (1/500, Jackson ImmunoResearch, 115-165-205) or anti-goat DyLight 550 (1/500, Diagomics, DKXGT-003) and Hoechst (1 µg/ml) in 2% DS for 45 min at RT, washed four times 10 min with PBS and mounted in PBS/Glycerol 75%. Images were taken with a Zeiss LSM800 confocal (×40 objective) and processed with Imaris 7.2.1 software (Bitplane).