Anti icam2 antibody

This protocol was adapted from a protocol described by Robins et al. (2013 (link)). Endothelial cells were isolated from lungs of 8 – 10-wk-old ${\text{apoE}}^{-/-}$ or DKO mice. The lungs were cut into small pieces and incubated at 37°C for 60 min in 0.1% collagenase A (Roche Diagnostics) in RPMI medium plus penicillin/streptomycin. The suspension was transferred into a $50–\mathrm{mL}$ tube, passed 15 times through an $18-\text{gauge}$ needle, and then through a cell strainer ( $70\mathrm{\mu }\mathrm{m}$ ), and was then centrifuged at $210\times \mathit{g}$ for 5 min. The supernatant was removed, and the cells were plated in $75{-\mathrm{cm}}^2$ tissue culture flasks coated with 0.1% gelatin (Millipore) and grown for 1 wk in 50% Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Wisent, Inc) supplemented with 10% FBS and penicillin/streptomycin and 50% Endothelial Growth Medium SingleQuot (EGM-2 SingleQuot) medium (Lonza). Endothelial cells were selected by performing two consecutive immunoisolation steps using magnetic beads conjugated with anti–intercellular adhesion molecule 2 (anti-ICAM-2) antibody (BD Biosciences PharMingen) and were plated into $75{-\mathrm{cm}}^2$ tissue culture–gelatin-coated flasks. Endothelial cells were used for experiments at passage 4. Cultures were serum-starved for 24 h before experiments.