| Protocol | Cell Assay: 1×104 HUVECs are plated per well of 96 well plate in EGM. After reaching sub-confluency cells are cultured in different concentration of Adalimumab (0.01-10 μg/mL), IgG isotype control (10 μg/mL) and 0.1% Triton-X as positive control in 5% CO2 at 37°C. After 12 hours, 10% Alamar blue is added to each well and kept in incubator for another 6 hours. Absorbance of oxidized alamar blue is measured at 600 nm on Infinite M200 pro plate reader. All measurements are performed in triplicate.
Animal Studies: In order to analyze the vascular distribution of Adalimumab, the substance is labelled using the DyLight 549 Microscale Antibody Labeling Kit. Briefly, one vial of DyLight 549 Reagent is dissolved in 1 mg Adalimumab solution (in PBS) and purified with a resin spin column to remove excess fluor. Hypercholesterolemic Ldlr-/- mice are injected with fluorophore-labelled Adalimumab (8.0 mg/kg in 200 μL PBS i.p.) for two consecutive days and sacrificed 12 hours after last injection. Vehicle injection (200 μL PBS) is used as control. |
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