Rabbit anti jam a igg

BrdU-labeled HuMEC-1 were placed on fibronectin-coated 96-well plates and left overnight at 37°C and 5% CO₂ to form an endothelial monolayer. HuMEC-1 and PBL were activated with LPS as previously described at 4°C. PBL and HuMEC-1 were co-cultured at 37°C for 120 minutes to simulate a rewarming process [9 (link)]. After removal of the PBL and washing of the HuMEC-1, the endothelial monolayer was fixed with 4% paraformaldehyde (AppliChem), followed by blocking with PBS supplemented with 10% donkey serum (Jackson ImmunoResearch).
Rabbit anti-JAM-A-IgG (1: 200; Santa Cruz Biotechnology) and goat anti-JAM-B-IgG (1: 100; Santa Cruz Biotechnology), donkey anti-rabbit-IgG-PE (1: 100; Santa Cruz Biotechnology) and donkey anti-goat-IgG-FITC (1: 100; Santa Cruz Biotechnology) in PBS containing 1% donkey serum were used for primary and secondary staining of the HuMEC-1, as previously described [9 (link)]. JAM surface protein expression was measured using a microplate reader at 485/535 nm and 530/590 nm, and the fluorescence intensity (FI) was calculated as a ratio to control. Each expression assay was performed in duplicates (PBL from 3 individual volunteers). To reduce cross-contamination of leukocytes, analysis was performed in a separate experiment independently from the transmigration assay.