Liberase

Primary cultures of murine articular chondrocytes were obtained from newborn mice as previously described^{31 (link)}. Briefly, joint cartilage was isolated from femur of 6-day-old mice. To separate chondrocytes from matrix, cartilage underwent 3 successive digestions: the first 2 digestions involved incubating the samples with liberase (Roche, Switzerland), 0.52 U/mL, for 45 min at 37 °C, then overnight with 0.13 U/mL liberase. The solution was passed through 25-, 10-, 5-, 2-ml Pasteur pipettes successively to break any aggregates. Then, the isolated cell suspension was filtered through a sterile 100-μM cell strainer and centrifuged at 2000 g for 15 min. The pellet was suspended in 3 mL DMEM with 10% fetal bovine serum (FBS) and seeded at 40000 cells per cm². Confluence was reached after 6 days of culture. Chondrocytes were pre-stimulated with interleukin 1 (IL-1) at 10 ng/mL for 24 h, washed and then cultured for 96 h with PBS, or with Furin 10 U/mL (P8077L, New England Biolabs); or with Furin inhibitor α1-PDX, 8 µM (RP-070 Thermo Scientific); and/or the TGFβ inhibitor SB431542, 50 µM (S4317, Sigma Aldrich France). Control chondrocytes were cultured in the presence of phosphate buffered saline (PBS) alone after seeding.

Cells from the intestinal LP were isolated as described previously^{4 (link),44} . Briefly, the tissues were digested with 1 mg/ml Collagenase VIII (Sigma–Aldrich) (SI) or 0.85 mg/ml Collagenase V (Sigma-Aldrich), 1.25 mg/ml Collagenase D (Roche), 1 mg/ml Dispase (Gibco) and 30 μg/ml DNase I (Roche) (colon). Peritoneal cells were isolated by lavage with 4 ml PBS/3% FCS. Mice were perfused with PBS and liver, spleen, lungs and brain were removed and macerated. Lungs were digested with 20 μg/ml Liberase (Roche) and 30 μg/ml DNase (Roche). Spleens and brains were digested with 1 mg/ml Collagenase D (Roche) and 0.15 mg/ml DNase. Liver was digested with 0.5 mg/ml Collagenase A (Roche) followed by Percoll (GE Healthcare) density centrifugation. The ventral and dorsal sheets of the ear were separated from the cartilage and incubated with 2.5 mg/ml Dispase (Gibco) to separate the epidermal and dermal sheets before incubation with 0.25 mg/ml Liberase (Roche) and 0.15 mg/ml DNase. Single cell suspensions were filtered through 100 μm strainers and resuspended in PBS/3% FCS for further analysis.

Thymic epithelia cells (TECs) were obtained from thymic epithelial tumors as previously described with minor modifications [28 (link), 29 (link)]. Thymic tissues were immediately placed in ice cold RPMI 1640 medium, cut in 1–3 mm³ pieces and transferred in "Liberase digestion solution" (RPMI 1640 medium supplemented with 0.5 U/ml Liberase (Roche) and 0.1% w/v DNase I) with ~ 2 ml of digestion solution/ cm³ of tissues. After 20 min incubation at 37°C under gentle agitation, supernatants were collected, mixed (v/v) with 1X PBS supplemented with 0.1% bovine serum albumin, and 0.5 mM EDTA, and centrifuged at 480g for 10 min at 4°C. The digestion procedure was repeated 4–6 times. Cells were counted in trypan blue using a Cellometer (Nexcelon BioSciences), seeded at 2–4.10⁶ cells/ cm² in "TEC medium" (RPMI 1640 medium supplemented with 2% Ultroser serum substitute (Pall corporation) and penicillin/streptomycin) and incubated at 37°C, 5% CO₂ in humid atmosphere. After 24 hours, cell culture supernatants were centrifuged at 480g for10 min at 4°C to eliminate non-adherent cells, supplemented with an equal volume of new "TEC medium" and added to the cultured cells. Cells were checked daily and passaged at confluence with trypsin EDTA solution.

Myeloid cells were isolated after enzymatic digestion of skin by 0.15 mg/ml liberase (Roche, Mannheim, Germany) and 0.12 mg/ml DNase (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) as described 31 (link). After 1 h myeloid cells were isolated using CD11b+ Cell Isolation Kit (Miltenyi, BergischGladbach, Germany) according to manufacturer´s instructions.
Epidermal cells were isolated upon segregation of the epidermis from dermis by overnight incubation with 0.25% trypsin (Biochrom AG, Berlin, Germany).
Dermal WAT was mechanically removed from the dermis. For isolation of SVF and dWAT dWAT was digested by 0.15 mg/ml liberase (Roche) for 1 h at 37°C. Tissue homogenate was filtered through a 70 µm filter and centrifuged at 4000 rpm for 10 min. The floating cell layer containing adipocytes was isolated and washed once. The cell pellet represents the SVF and was washed once with PBS.