Fluorescent spectrophotometer

Whole cell lysate were collected following treatment with RIPA buffer (Millipore, MA) with addition of protease inhibitor (Roche, Laval, QC, Canada). The total protein concentrations of the samples measured using a commercially available BCA protein assay kit (Pierce, Rockford, IL, USA). Enzyme assay for SIRT1 activity was performed as per the manufacturer instructions (Sigma-Aldrich). The plates were read with a fluorescent spectrophotometer (Biotek, Winooski, VT, USA) at excitation 340 nm and emission 430 nm.

Human microvascular endothelial cells were seeded onto inserts (1 μm pores) in 24-well plates, with or without incubation with specific reagents for 24 hrs, and were tested for vascular permeability using the In Vitro Vascular Permeability Assay Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Endothelial cells were seeded at a concentration of 2 × 10⁶ cells/ml (200 μl/insert). Cell morphology and confluency was monitored under the microscope and upon formation of monolayer following 72 hrs, cells were treated with specific reagents (Ad-SIRT1, p300 siRNA, p300 plasmid, SIRT1 siRNA) with or without glucose. Following treatment FITC-Dextran is added to the cells and permeability of this fluorescent molecule is measured at several intervals (0.5, 1, and 2 hrs) by measuring the fluorescence of the receiver plate well solution. The plates were read with a fluorescent spectrophotometer (Biotek) at excitation 485 nm and emission 535 nm.