Igepal ca 630 pbs

Livers were perfused with ice-cold PBS and minced on ice followed by enzymatic digestion in IMDM (Gibco, Germany) containing 0.2 mg/ml collagenase D (Roche, Germany), 10 μg/ml DNase (Sigma, Germany) and 5% FCS for 30 minutes at 37 °C. After addition of EDTA (5 mM final concentration), cells were pelleted by centrifugation (15 minutes at 300× g) and liver lymphocytes were enriched by Percoll density gradient centrifugation. The cells were stimulated with a mixture of OVA protein and the OVA peptides CD4_323–339 and CD8_257–264 (20 μg/ml final concentration for all) for 24 hours at 37 °C. The cells were then stimulated by incubation with 0.01 μg/ml PMA (Sigma, Germany) and 1 μg/ml ionomycin (Sigma, Germany) for a total of 4 hours, 10 μg/ml Brefeldin A (Sigma, Germany) were added after 2 hours. For flow cytometric analysis, Fc-block was performed through incubation with anti-mouse CD16/CD36 antibody (2.4G2) followed by surface staining for mouse CD8 (53–6.7) and CD44 (IM7) (BD Biosciences, eBioscience). After fixation (2% paraformaldehyde/PBS v/v), cells were permeabilized using 0.1% Igepal^® CA-630/PBS (Sigma, Germany) and stained for intracellular IFNγ (XMG1.2). Data were acquired on an LSR Fortessa instrument (BD Biosciences) and further analysed using the FlowJo software (Tree Star, USA).

Synovial tissue extracts were prepared as described previously by Rosengren et al. [70 (link)]. The left knee joints (5 mm² size) were collected, placed on ice, and minced to smaller fragments with a scalpel. The fragments were weighed, snap-frozen in liquid nitrogen, and stored at −80 °C until the time of extraction. Ice-cold extraction buffer, consisting of 0.1% Igepal CA-630/PBS (#I8896, Sigma-Aldrich, Munich, Germany) with protease inhibitors (#A32963, Pierce Protease Inhibitor Tablets, Thermo Fisher Scientific, Waltham, MA, USA), was added at a volume of 100 μL per 1 mg of tissue. The samples were prepared on ice using a cordless micro-tube homogenizer (#BAF651000000; SP Bel-Art^® Proculture Homogenizer system; Sigma-Aldrich, Munich, Germany) and were disintegrated until only white fibrous insoluble connective or bone tissue remained. After incubation for 20 min on ice, the samples were subjected to centrifugation for 10 min/200× g at 4 °C. The supernatant was collected, aliquoted, and stored at −80 °C for downstream analysis.