Ms minimacs separation columns

Livers were perfused with 50 mL saline solution, minced, suspended in 5 mL Hank’s Balanced Salt Solution (HBSS) (20 µg/mL collagenase, 150 U/mL DNase I, and 20 mM HEPES, pH = 7.4), and incubated for 20 min at 37 °C in a gentleMACS™ Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cell suspension was filtered through a 40-µm nylon mesh and centrifuged at 300 × g for 6 min. Cell pellets were suspended in Percoll (final concentration, 35% for leukocyte isolation; 33% for Kupffer cell isolation), and centrifuged at 370 × g and 500 × g, respectively, for 10 min. Cell pellets were washed once with PBS, resuspended in PBS and the cell number was counted with haemocytometer. More than 90% of the resulting cells were alive according to trypan blue exclusion. For Kupffer cell isolation, CD11b⁺ cells were isolated from the cells using anti-CD11b microbeads over MS⁺ MiniMACS separation columns (Miltenyi Biotec) according to manufacturer’s instructions. Most purified cells were alive (>90%, trypan blue exclusion) and were >90% CD11b⁺ cells.

Mouse LECs were isolated from non-treated mice using a previously described method with modifications^{36 (link)}. In brief, lungs were removed from untreated WT or Spred2^−/− mice. They were minced in a gentleMACSTM Dissociator (Miltenyi Biotec, Gladbach, Germany) and digested in 5 ml Hank’s balanced salt solution (HBSS) containing 20 μg/ml collagenase (Wako, Japan), 150 U/ml DNase I (Roche, Basel, Switzerland) and 20 mM HEPES (Gibco), for 30 min at 37 °C with occasional shaking. The cell suspensions were filtered through 100-μm nylon mesh, and centrifuged at 400 × g for 6 min. The cells were washed once with PBS and then incubated with anti-CD45 microbeads. CD45⁺ cells were removed over MS MiniMACS separation columns (Miltenyi Biotec), and CD45⁻ cells were collected and incubated with biotin-labeled anti-CD326 Ab (Biolegend, USA), followed by addition of streptavidin-coated microbeads. Magnetic beads-labeled CD326⁺ cells were collected on a separation column, suspended in DMEM high glucose medium containing 10% FBS. CD326⁺ cells included bronchial and alveolar epithelial cells.