Primary pulmonary microvascular endothelial cells (PMVECs) were isolated from lung tissues in DHCR24 flox/flox-Tie2Cre− mice and DHCR24 flox/flox-Tie2Cre+ mice, as previously described (26 (link)). Hundred microliter of sheep anti-rat IgG Dynabeads (Thermo Fisher, USA) were mixed with 5 µg anti-mouse PECAM-1 (BD Pharmingen, USA). The beads and antibody were incubated overnight at 4°C. Fresh mouse lung tissue was isolated and washed with PBS, before being transferred to cryopreservation tubes and cut into pieces with sterile scissors. The lung tissue fragments were digested with collagenase for 45 minutes in a 37°C shaker. The cells were filtered through sterile 70-μm nylon mesh and washed twice with 0.1% bovine serum albumin (BSA; Solarbio, China).
To allow binding to cells, 30 µL of coated Dynabeads were added to cells and incubated for 25 minutes at room temperature. All bead-bound cells were resuspended in DMEM containing 20% FBS before plating and purified with Anti-ICAM-2 (BD Pharmingen) antibody-conjugated Dynabeads when the cell confluences reached 70% to 80% or above.
To allow binding to cells, 30 µL of coated Dynabeads were added to cells and incubated for 25 minutes at room temperature. All bead-bound cells were resuspended in DMEM containing 20% FBS before plating and purified with Anti-ICAM-2 (BD Pharmingen) antibody-conjugated Dynabeads when the cell confluences reached 70% to 80% or above.