Fitc conjugated bs 1 lectin

The LEPCs were isolated and cultured according to the previous description [24 (link)]. The human-derived peripheral blood mononuclear cells (PBMCs) were inoculated into 6-well plates containing 5 μg/mL human fibronectin, followed by culturing in the EGM-2 medium (Promocell, USA). The cells were cultured at 37 °C with 5% CO₂ in a humid environment. After 4 days of culture, the cells were washed with PBS to remove the non-adherent cells, and the original medium was replaced. Typically, EPCs were positive in the uptake of 0.02 mg/mL DiI-acLDL (Invitrogen, USA) and binding of 0.01 mg/mL FITC-conjugated BS-1 lectin (Sigma-Aldrich, USA), according to the previous description [25 (link)]. Also, flow cytometry was done to examine the endothelial markers in the cultivated LEPCs using the following antibodies: 1. FITC-conjugated monoclonal mouse anti-human antibodies that recognize CD31 (eBioscience, USA) and vWF (Novus Biologicals, USA), and 2. APC-conjugated monoclonal mouse anti-human antibodies that recognize Tie-2 (Novus Biologicals, USA). Notably, LEPCs at the third passage (approximately 4 weeks) were utilized in each of the assays. According to the separation and culture protocol, EPCs were the cells that attached and appeared spindle-shaped [26 (link)].

This assay was performed according to the protocol of Baker and Robinson^{54 (link)}. In summary, thoracic aortae were dissected from mixed background mice, cut into rings approximately 0.5 mm in width and incubated in serum-free Opti-MEM (Gibco, cat. no. 51985026) at 37 °C overnight. Each ring was then embedded in separate wells of a 96-well plate containing 1.2 mg/ml of collagen I (Millipore, cat. no. 08115), which was polymerised by leaving the plate at 37 °C for 30 mins. Rings were fed with fresh Opti-MEM supplemented with 2.5% FBS and VEGF (30 ng/ml) at 37 °C every 3 days with different doses of SFN. After 6 days, rings were fixed with 4% (v/v) formalin and the microvessel counted by phase-contrast microscopy. To further staining, specific epitopes can be fluorescently labelled. After the fixing, the rings were permeabilised with 0.25% (v/v) Triton X-100 in PBS, and blocked with 2% (v/v) BSA in PBLEC (PBS with 1 mM CaCl₂, 1 mM MgCl₂, 0.1 mM MnCl₂, 1% Tween-20) for 30 mins at 37 °C. FITC-conjugated BS-1 lectin (Sigma, cat. no. L9381/L5264) was used to stain endothelial cells and anti-actin α-smooth muscle Cy3 (α-SMA; Sigma, cat. no. C6198) was used to stain the supporting cells. 1 µg/ml DAPI was used to stain the nuclei. Images were taken by Axiovert 40 CFL inverted microscope (Carl Zeiss).