Fluorescein isothiocyanate fitc labeled dextran

In vitro endothelial monolayer permeability assay was performed according to previously described methods [22 (link)]. HBMECs were seeded on the inner surface of collagen-coated transwell inserts (6.5mm diameter, 0.4μm pore size polycarbonate filter; Corning, Corning, NY), which were placed in wells of a 24-well plate with complete EBM-2 media. When the monolayer of cells was confluent, confirmed by ensuring that it is impermeable to media, the cells were starved for 8h with EBM-2 media without growth supplement and serum before treatment. The cells were treated with 50ng/ml of TNFα with or without 1μg/ml of LCN2 for 24hr before permeability measurement. Permeability was measured by adding 0.1 mg/ml of Fluorescein isothiocyanate (FITC)-labeled dextran (MW, 70,000; Sigma, St. Louis, MO) to the upper chamber, with lower compartment containing fresh serum-free media. After incubation for 20 min, 100μl of sample from the lower compartment was measured for fluorescence at excitation 490nm and emission 520nm. All independent experiments were performed in duplicate or triplicate.

For intravital microscopy, mice fasted overnight were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively, intraperitoneally). After laparotomy, the left liver lobe and epididymal fat were externalized for assessment of microcirculation using intravital microscopy (Olympus BX150WI; Center Valley, PA, USA), as described previously [17 (link)]. After intravenously administering 0.3 mg/kg rhodamine 6G (Sigma Chemical Co., St. Louis, MO, USA), leukocyte-endothelial interaction was assessed by counting the number of labeled leukocytes rolling or adhering to hepatic or adipose tissue microcirculation. In the liver, the number of vitamin A-positive hepatic stellate cells (HSCs) was determined as the number of fluorescent cells derived from vitamin A autofluorescence. After intravenously administering 0.05 mL of 2% fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight 150,000; Sigma Chemical Co., St. Louis, MO, USA), hepatic microcirculation images were acquired (Prime Intervision, Doral, FL, USA). Sinusoidal density analysis was performed using ImageJ software (ImageJ 1.47 v; Wayne Rasband, National Institute of Health, Bethesda, MD, USA) to determine the mean value of functional capillary density.

Purified Stx2a was provided from Phoenix Laboratory (Tufts Medical Center, Boston, MA, USA). Eliglustat (Cerdelga, Sanofis-Genzyme) used as an inhibitor of glucosylceramide synthase (Shayman, 2013 (link)) was purchased from MedKoo Biosci, Morrisville, USA. Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., 2006 (link)), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, 2007 (link)), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., 2010 (link)) were purchased from Sigma Aldrich, St. Louis, MO, USA. A fluorescein isothiocyanate (FITC)-labeled Dextran (average molecular weight of 70 kDa, Sigma Aldrich, catalog # 46945) was used as a marker of paracellular permeability (Chattopadhyay et al., 2017 (link)). A mouse monoclonal antibody against the A-subunit of Stx2 (Mab 2E11) was provided by Roxane Piazza (Butantan Institute, São Paulo, SP, Brazil) (Rocha et al., 2012 (link)) and an Alexa 647-conjugated anti-mouse secondary antibody (AbCam, catalog #ab150115) were used for flow cytometry.

Mice were administered 0.6 mg/kg fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight, 4,000; Sigma-Aldrich) in PBS by oral gavage. Four hours later, blood samples were collected by cardiac puncture, and the fluorescence was measured using a Synergy H4 Hybrid Reader (Biotek, VT, USA).

1 × 10⁵ Eph4 cells were seeded onto the transwell inserts (cellulose mixed ester, 24-well, pore size 0.4 μm, translucent, BD Falcon), and cultured for four days. After TER values had reached a plateau, media were changed from D-MEM high glucose to Opti-MEM (Thermo Fisher Scientific), and 50 μg of mouse IgG or the mAb 67-2 were added to the lower chamber. 2 mM EGTA (Sigma) was applied to the upper chamber. TER value was measured with cellZscope instrument (CellSeed, Tokyo, Japan).
For the paracellular tracer flux assay, 10 min after applying mouse IgG, 67-2 or EGTA, 1 mg/ml fluorescein isothiocyanate (FITC)-labeled dextran with the molecular mass of 3-4 kDa, 70 kDa and 250 kDa (Sigma) was added to the upper chamber. After incubation for 2 h, the concentration of each tracer in the lower chamber was determined by measuring fluorescence with microplate reader (Varioskan, Thermo Fisher Scientific).