Single cell suspensions from thoracic aorta were prepared by enzymatic digestion as previously described with minor modifications (36 , 37 (link)). Briefly, thoracic aorta segments were dissected following vasculature perfusion with 2mM EDTA buffer(5ml), PBS (10ml), and FACS buffer (10ml) respectively. Thoracic aorta, segment above diaphragm, was cut into small pieces and digested in 3ml enzyme cocktail containing 400 U/ml collagenase type I (Millipore Sigma, #C0130), 120 U/ml collagenase type XI (Millipore Sigma, #C7657), 60 U/ml hyaluronidase type I (Millipore Sigma, #H3506), and 60 U/ml DNase I (Millipore Sigma, #11284932001) in RPMI supplemented with 10% heat-inactivated fetal bovine serum (hiFBS) for 50 minutes at 37°C. Digested products in suspensions were filtered through a 40 μm cell strainer (BD Falcon, San Jose, CA) followed by red blood cell (RBC) lysis (ACK lysis buffer, Millipore Sigma) for 3 min at room temperature.
Single cell suspensions from mouse lung, draining lymph nodes, or spleen were prepared by mechanically mincing collected organs though a 40 μm strainer followed by RBC lysis for 3 min at room temperature as described (38 (link)).
Single cell suspensions from mouse lung, draining lymph nodes, or spleen were prepared by mechanically mincing collected organs though a 40 μm strainer followed by RBC lysis for 3 min at room temperature as described (38 (link)).
