Alexa fluor 594 phalloidin

Cells were seeded onto 0.17 mm coverslips and immunofluorescence was performed as described²⁸ , briefly, coverslips containing cells were washed twice with PBS, followed by incubation with 250 ul of BD Cytofix/Cytoperm solution for 20 minutes at 4 C. Anti-CD-63 antibody (FITC-conjugated) was diluted in BD Perm/Wash buffer at a concentration of 1:250 when used for staining CD63 alone, or with Alexa Fluor™ 594 Phalloidin (5 um final concentration), when used for simultaneous detection of CD-63 and F-actin and incubated on ice for 30 minutes. After which coverslips were washed twice with PBS, mounted with antifade reagent containing DAPI and observed under confocal microscope.
For exosome uptake assay by HUVECs, exosomes isolated from THP-1 cells were labeled with DiO-C16 for 1 hour and then unlabeled dye was removed by washing with PBS. Purified labeled exosomes were isolated by floating DIO labeled exosomes on sucrose density gradient. THP-1 exosomes thus isolated were resuspended in M-199 medium and incubated with cultured HUVEC cells. After incubation for 4 hours, HUVEC cells were washed, fixed, and observed under confocal microscopy.

ECs were grown to confluence on glass coverslips under normal growth conditions and treated with L-NIO (5 mM) ± [NTG (5 μM) or NTG-NL (5 μg/ml)] for 24 hr. Next, a monocyte adhesion assay was performed (as described earlier) and the U937 cell-EC cocultures fixed, permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA; Millipore, USA), and sequentially incubated with primary anti-ICAM-1 mouse antibody (Santa Cruz Biotechnology, USA) and secondary FITC-conjugated DyLight 488 anti-mouse IgG (Vector Labs, USA). To visualize actin microfilaments, U937 cell-EC cocultures were incubated with Alexa Fluor 594-Phalloidin (BD Biosciences, USA). Coverslips were mounted onto glass slides and fluorescence images (15 per condition) were taken using a Nikon Eclipse Ti microscope fitted with a Nikon Digital Sight DS-Qi1Mc camera. ICAM-1 clustering index was determined by measuring ICAM-1 fluorescence intensity (from n ≥ 10 images) at the U937-EC adhesion site and normalizing it to the average ‘background’ intensity measured from three neighboring EC cytoplasmic sites.