Imaging Endothelial Glycocalyx Components

To image the endothelial glycocalyx, an Alexa Fluor 594-conjugated wheat germ agglutinin (WGA, Thermo Fischer Scientific, W11262, dilution 2 μg/ml), anti-heparan sulfate antibody (Abcam, Cambridge, UK, ab23418, 1:100), and peanut agglutinin (PNA, Vector Labs, Ontario, CA, FL-1071-5, 1:200) were used. Cells were cultured to confluence on coverslips and exposed to cyclosporine as described. Cells exposed to 500 mU/mL neuraminidase for 1 h were used as a positive control in WGA and PNA experiments. Cells exposed to 0.5 U/mL Heparinase III (H8891-5UN, Sigma-Aldrich, St. Louis, MO) for 30 min were used as a positive control in heparan sulfate experiments. Cells were incubated with Alexa Fluor 594-conjugated WGA for 5 min on ice and washed two times with ice-cold HBSS, and the coverslips were mounted in a Chamlide magnetic chamber (Life Cell Instrument, Seoul, Korea) and overlaid with media. Confocal microscopy was performed as detailed in Supplementary material, and total fluorescence intensity was measured using ImageJ software. For experiments using anti-heparan sulfate and PNA, cells were washed and fixed with 2% paraformaldehyde, followed by incubation with mouse anti-heparan sulfate (1:100) and anti-PNA (1:100) for 1 h. Alexa Fluor 488-conjugated species-specific secondary antibodies were used at a dilution of 1:1,000. Nuclei of cells were stained with 0.12 μg/ml Hoechst stain (Thermo Fisher Scientific, Waltham, MA) for 5 min.