Collagenase 2

The stromal vascular cells (SVCs) were isolated from ATs and splenocytes were isolated from spleen as previously described (43 (link)). In brief, tissues were dissected and mechanically minced for digestion with 2 mg/mL collagenase II (Sigma Aldrich) for SVCs and 1 mg/mL collagenase II for splenocytes at 37°C for 30 min with shaking. Digested cells were then filtered and centrifuged at 500xg 4°C for 5 min. Subsequently, cells were undergoing RBC lysis for 5 min at RT. Cells were then counted and incubated with CD16/CD32 antibody for 10 min at 4°C and stained with indicated antibodies for 30 min at 4°C. For Tregs, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBiosciences), then further stained with Anti-Mouse Foxp3 antibody for 2 h at 4°C. Flow cytometry analyses were performed on BD FACS Canto™ II (Beckon Dickinson) and analysed with FlowJo software (Treestar). All antibodies were listed in Supplementary Table 1.

Adult cardiac fibroblasts were isolated from 6–8 weeks old C57BL/6J male mice. The hearts were perfused using a Langendorff-Free method [27 (link)]. Hearts were removed followed by injection into the apex of left ventricle with 7 ml of EDTA buffer containing 130 mM NaCl, 5 mM KCl, 0.5 mM NaH₂PO₄, 10 mM Glucose, 10 mM 2,3-butanedione monoxime (BDM), 10 mM Taurine, 5 mM EDTA and 10 mM HEPES, pH 7.8. Then the ascending aorta was clamped using surgical hemostats, and the heart was transferred to a fresh EDTA buffer This was followed by injection, at 2 ml/min, of 10 ml EDTA buffer, then 3 ml perfusion buffer containing 130 mM NaCl, 5 mM KCl, 0.5 mM NaH₂PO₄, 10 mM Glucose, 10 mM BDM, 10 mM Taurine, 1 mM MgCl₂ and 10 mM HEPES, pH 7.8, and 50 ml collagenase buffer containing 0.5 mg/ml Collagenase 2 (Sigma-Aldrich C6885); 0.1 mg/ml Collagenase 4 (Sigma-Aldrich C5138); Protease type XIV, 0.05 mg/ml (Sigma-Aldrich P5147) all dissolved in a perfusion buffer. Cells were dissociated with gentle trituration, and digestion was stopped by addition of 5 ml of perfusion buffer containing 5% FBS. Cells were passed through a 100 μm cell strainer, centrifuged, washed in DMEM (Gibco, 11995) containing 10% FBS and resuspended in 10% FBS DMEM, 10 U/ml Penicillin, and 100 μg/ml Streptomycin and plated onto 10 cm culture dishes (Corning, 430167).

Immunotyping of the PDL tissue by flow cytometry (FACS) analysis was performed as previously described (22 (link)). Prior to OTM and following anesthesia, 21 mice (n=7/group) were divided into 3 groups: (a) experimental group which received a single sub-gingival injection of 10μl of 0.1 mg/ml RvD1 (b) control (sham) group which received a single sub-gingival injection of 10μl saline (c) control group with inactivated springs and no subgingival injections. Mice were sacrificed after 24 hours and the ULM1 teeth were gently extracted and incubated in working solution containing PBS (x1), 2% Fetal Calf Serum (Sigma Aldrich), Collagenase 2 (1μg/ml; Sigma Aldrich) and DNase (1μg/ml; Sigma Aldrich) for 25 minutes at 37°C on a shaker. Following incubation, EDTA 0.5M was added to working solution. The solutions were collected, filtered and centrifuged (1400 rpm; 8 minutes; 4°C). Aliquots of the samples were divided into experimental groups and stained with fluorochrome-conjugated monoclonal antibodies: CD45, CD3, Ly-6G, CD8, F4/80, CD64, CD265 (BioLegend^®) for the detection of leukocytes, T cells, neutrophils, T cytotoxic cells, macrophages, monocytes and Receptor Activator of Nuclear Factor κB (RANK); respectively. Following incubation, samples were analyzed using the BD LSR Fortessa™ cell analyzer.

For the analysis of lungs, BALF was obtained by flushing lungs twice with 0.7 ml PBS using a tracheal cannula (Terumo, SURFLO, 20 G). Then, the left lung lobe was fixed in 4% paraformaldehyde (Bioworld) for histological analysis. The right lung lobes were minced and digested with 50 μg/ml of Liberase-TM (Roche) and 20 ng/ml of DNaseI (D5025, Sigma-Aldrich, 750 Kunits/mg) in RPMI 1640 Medium (ThermoFisher) for 1 h at 37 °C, then mashed through 40 μm cell strainers to obtain single cell suspension. For the lysis of red blood cells in BALF and lung single cell suspension, ACK lysing buffer (Lonza) was used. Then, isolated cells were stained and analyzed by FACS.
For the analysis of lymph nodes, EDLN was chopped and digested with 1 mg/ml of Collagenase 2 (C6885, Sigma-Aldrich) and 40 ng/ml of DNaseI in RPMI 1640 Medium for 30 min at 37 °C, then filtered through 70 μm cell strainers. Isolated cells were then analyzed by FACS.
For the analysis of ear tissue, ear was minced and digested with 50 μg/ml of Liberase-TM and 20 ng/ml of DNaseI in RPMI 1640 Medium for 1 hr at 37 °C, then mashed through 40 μm cell strainers to obtain single cell suspension. The isolated cells were subsequently analyzed by FACS.

Murine gonadal AT were enzymatically digested with 1 mg/ml Collagenase D (Roche) for 35 min at 37 °C in RPMI 1640 (Sigma) containing 1% fetal bovine serum (Sigma). Peritoneal exudate cells were isolated by flushing murine peritoneal cavities with RPMI 1640 (Sigma). The liver was perfused before dissection with 5 ml of RPMI 1640 (Sigma) injected through the portal vein. The tissue was cut into small pieces and homogenized using the gentleMACS dissociator (Miltenyi) in buffer containing Collagenase 2 (Sigma 0.425 mg/ml), Collagenase D (Roche 0.625 mg/ml), Dispase (Gibco 1 mg/ml), and DNase (Roche 30 µg/ml). After 20-min incubation at 37 °C, the tissue was homogenized further using the dissociator. Red blood cells were lysed using red blood cell lysis buffer (Sigma).