Enzymatic Tissue Dissociation Protocols

All enzymes were purchased from Sigma-Aldrich, unless stated otherwise. Tissues were isolated in 5% FBS (Gibco) in RPMI-1640 (Gibco). Tissues were cut into small pieces and digested in the appropriate enzyme mixture at 37 °C in a water-bath shaker: Aorta:^{59 (link)} 450 U/ml collagenase I, 125 U/ml collagenase XI 60 U/ml hyaluronidase and 60 U/ml DNase I (ThermoFisher Scientific) for 50 min; Lung: 0.4 mg/ml collagenase IV and 0.15 mg/ml DNase I for 40 min; Heart: 1 mg/ml collagenase II and 0.15 mg/ml DNase I for 30 min. The cells were retrieved by passing tissue pieces through a 70 μm cell strainer (Greiner Bio-One). Spleen and lymph nodes were mechanically dissociated by passing cells through a 70 μm cell strainer. For separating aortic layers into the intima/media and the adventitia, the whole aorta was pre-incubated with 125 U/ml collagenase II and 3.75 U/ml elastase at 37 °C for 10 min. The adventitia layer was separated from the inner tube (the intima and media) by pulling with two pairs of fine forceps. The tissues were then digested as performed in the aorta sample. For lungs, hearts, and spleens, erythrocytes were lysed using Red Cell Lysis Buffer (Sigma-Aldrich) at room temperature for 2 min.