Mouse lung EC were isolated from three to four pairs of lungs dissected from control or Sptlc1 ECKO adult mice 4 weeks post tamoxifen treatment. Freshly isolated lung tissues were minced with scissors and allowed to digest at 37 °C in Liberase (0.6 U/mL, Sigma) in HBSS for 45 min. Samples were further subjected to mechanical disruption by gentleMACS Octo Dissociator (Miltenyi Biotec) and filtration through fine steel mesh (40 µm, Falcon). Cells were washed twice with HBSS containing 0.5% Fatty Acid Free BSA. RBC were removed by ACK lysis buffer (Thermo Fisher). Cells were incubated with CD45 micro beads (Miltenyi Biotec) at 4 °C for 15 min to deplete CD45+ cells. Remaining cells were incubated with CD31 micro beads (Miltenyi Biotec) at 4 °C for 15 min to enrich EC. The purity of each population was determined by flow cytometry.
Cells were stained with stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (BioLegend, 1:200), allophycocyanin (APC)-conjugated anti-mouse CD45 (BioLegend, 1:200) in blocking solution (0.25% FAF-BSA in HBSS) with anti-CD16/32 (BioLegend, 1:200) for 15 min on ice. Stained cells were washed with HBSS containing 10% serum twice and analyzed by BD FACSCalibur Flow Cytometer (BD Biosciences). CD31 and CD45 were gated as shown inFigure 1B .
Cells were stained with stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (BioLegend, 1:200), allophycocyanin (APC)-conjugated anti-mouse CD45 (BioLegend, 1:200) in blocking solution (0.25% FAF-BSA in HBSS) with anti-CD16/32 (BioLegend, 1:200) for 15 min on ice. Stained cells were washed with HBSS containing 10% serum twice and analyzed by BD FACSCalibur Flow Cytometer (BD Biosciences). CD31 and CD45 were gated as shown in