Cell imaging software

First differentiated podocytes grown on glass coverslips were fixed with paraformaldehyde (4% in PBS) and permeabilised with Triton ×-100 (0.25%). Then the cells were incubated with primary antibody directed against GSDMD-N (1:200) at 4°C overnight. Next, the coverslips were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:200) as a secondary antibody for 1 h at room temperature. Finally, the coverslips were mounted with Fluoroshield™ and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; F6057, Sigma-Aldrich, blue fluorescent dye) for 10 min in the dark. The images were acquired using an epifluorescence inverted microscope (IX81, Olympus, Tokyo, Japan) equipped with a cell imaging software (Soft Imaging System GmbH, Munster, Germany). The results were confirmed by a professional pathologist.

Paraformaldehyde-fixed cells from the phagocytosis assay were centrifuged at 200 g for 5 min and resuspended in PBS containing 0.1% Tween 20 and 2.5 µg. mL⁻¹ TRITC-conjugated phalloidin (Sigma-Aldrich). Cells were incubated with phalloidin for 40 min at room temperature, protected from light. Excess phalloidin was removed by centrifugation and cells were resuspended by pipetting up and down in buffered glycerol containing 0.5 µg/mL DAPI (Sigma-Aldrich). Cells were then visualized on a slide in a BX51 fluorescence microscope with a U‐UCD8 condenser, a U‐LH100HGAPO burner, a U‐RFL‐T power supply, a 63X/1.4 NA oil objective and a 450–490 nm excitation/500–550 emission bandpass filter (Olympus, Tokyo, Japan). Images were collected with a CC12 digital color camera and the Cell Imaging Software (Olympus) and composite figures were prepared using ImageJ (Schneider et al., 2012 (link)) and Photoshop CS5 (Adobe Systems, San Jose, CA) software.

Freshly excised synovial tissue samples (2–4 mm diameter fragments) from 10 OA patients and 8 RA patients were embedded in fibrin scaffolds in individual wells of 24-well culture plates (Corning, Corning, NY). Fibrin matrices were prepared as described previously with minor modifications (Rüger et al., 2008 (link)). In brief, human fibrinogen (2 mg/ml; Calbiochem, Darmstadt, Germany) was dissolved in PBS supplemented with 200 U/ml aprotinin (Gerot Pharmaceutica, Vienna, Austria) to prevent fibrinolysis. Human plasma thrombin (0.6 U/ml, Sigma) was added to the fibrinogen solution and gel formation occurred by incubation at 37°C for 30 min. The tissue fragments were cultured using complete M199 medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences), 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (Sigma) without additional growth factors for up to 4 weeks. The culture medium was changed every 3–4 days. The egress of cells into the fibrin matrix, cell growth and structural re-organization including vascular tube formation were monitored using a phase contrast microscope (Olympus IMT-2, Tokyo, Japan) and documented using a digital camera (Olympus DP50). The length of vascular sprouts was assessed in tissue fragments of four OA and RA patients, respectively, after 3 weeks of culture using the cell^* Imaging Software (Olympus).