Hyaluronidase type 4 s

In the laboratory, fragment of aneurysm was divided into two parts: wall (containing mostly intima-media) and PVT (containing PVAT and contiguous remodeled adventitia). Samples, were subsequently mechanically disrupted and digested with a cocktail of enzymes containing 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type IVS and 450 U/ml collagenase type I (all from Sigma-Aldrich, Irvine, UK) in PBS with calcium- magnesium containing 20 mM Hepes at 37°C for 20 min with gentle agitation to isolate residual cells infiltrating tissues. The resulting cell suspension was then passed through a 70 μm strainer (BD Pharmingen, San Diego, CA, USA). Cells were incubated with fluorescently labeled antibodies for 20 min at 4°C (for details see Supplemental Table 1). Fluorescence Minus One (FMO) was used as negative control. After washing, cells were re-suspended in PBS with 1% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, UK) and data acquired on a FACSCanto II cytometer (BD Bioscience, UK) and analyzed using FACSDiva™ and FlowJo software (Tree Star Inc, Olten, Switzerland).

Full-depth articular cartilage was excised and immersed in Dulbecco’s Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) (10 mM), penicillin (100 U/mL) and streptomycin (0.1 mg/mL) (all from Lonza, Belgium). After three washes, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/mL hyaluronidase type IV S (Sigma-Aldrich, Belgium) for 30 min at 37°C, 1 mg/mL pronase E (Merck, Belgium) for 1 h at 37°C and 0.5 mg/mL clostridial collagenase IA (Sigma-Aldrich, Belgium) for 16–20 h at 37°C. The enzymatically isolated cells were then filtered through a nylon mesh (70 µm), washed three times and counted.

Subcutaneous matrigel plugs were digested for 60–90 minutes at 37 °C in an enzymatic cocktail composed of 25 μg/ml DNase-I, grade II (Roche, cat no. 10-104-159-001), 3 U/ml Dispase (Roche, cat no. 10-269-638-001), 3 U/ml Liberase TM Research grade (Roche, cat no. 05-401-119-001) and 25 μg/ml Hyaluronidase Type IV-S (Sigma, cat no. H3884). The isolated cells were filtered through a 70 μm Nylon cell strainer (BD Falcon, Bedford, MA) into a sterile 50 ml centrifuge tube and cells washed 3X with FACS Buffer (1X PBS without Ca/Mg, 2 mM EDTA and 0.5% BSA).
Isolated cells were analyzed by flow cytometry to determine their cellular characteristics. FITC-conjugated F4/80 (eBioscience, cat no.11-4801) and APC-conjugated CD31 (eBioscience, cat no. 17-0311) were used to distinguish endothelial cells from macrophages with additional labeling using eFluor450-conjugated CD34 (eBioscience, cat no. 48-0341), PE-conjugated CD-133 (eBioscience, cat no. 12-1331) or PERC-P conjugated CD45 (eBioscience, cat no. 45-0451). Antibodies were incubated with isolated cells in FACS buffer for 30 minutes at 4 °C in darkness. Cells were washed 3X with FACS buffer and fixed in 1% PFA in PBS for 10 minutes at room temperature in the absence of light. Fixed cells were washed in PBS and resuspended in 400 μl of FACS buffer for flow cytometry. Experiment performed three times.

For the evaluation of infiltration of immune cells into hearts post-MI, the excised hearts were minced with fine scissors into 1–2 mm³ pieces. The minced heart tissue was transferred into a tube containing enzyme solution (200 units/mL collagenase II (Worthington, Lakewood, NJ, USA), 500 units/mL hyaluronidase type IV-S (Sigma-Aldrich, St. Louis, MO, USA), and 100 units/mL DNase I (Takara Biotec, Kusatsu, Japan) and dissociated using a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The dissociated cells were stained directly using fluorochrome-conjugated mouse-specific antibodies and analyzed with a FACSVerse™ instrument (BD Biosciences, San Jose, CA, USA) using FlowJo version 10.2 software (BD Biosciences). Cells that did not stain with 7-AAD (TONBO Biosciences, San Diego, CA, USA) were deemed viable. The antibodies used were as follows: anti-CD45 (clone 30-F11; eBioscience, San Diego, CA, USA), anti-Ly6G (clone RB6-8C5; TONBO Biosciences), anti-CD11b (clone M1/70; eBioscience), anti-F4/80 (clone BM8.1; TONBO Biosciences), and anti-CD206 (clone C068C2; BioLegend). To evaluate apoptosis in vitro, HL-1 cells were stained with 7-AAD and FITC Annexin V (BD Biosciences).