Anti cd105

After expansive culture, the cells were harvested by a treatment with EDTA/tripsyn, centrifuged at 1500 rpm for 5 min and re-suspended in Phosphate-Buffered Saline (PBS) (Sigma Aldrich, Darmstadt, Germany) and 0.2% Bovine Serum Albumin (BSA) was added (Sigma-Aldrich). The cells were then labeled for 15 min at room temperature (RT) in the dark with 5 μL of the following primary antibodies: Fluorescein isothiocyanate (FITC)-conjugated anti-CD14, anti-CD45, and anti-CD105 (Santa Cruz Biotechnologies, Dallas, TX, USA); Phycoerythrin (PE)-conjugated anti-CD29, anti-CD44 and anti-CD90 (BioLegend, San Diego, CA, USA). The negative controls were prepared by labeling cells with 5 μL of FITC- and PE-conjugated isotypic antibodies (Santa Cruz and BioLegend) for 15 min at RT in the dark. The data for 10,000 total events were acquired by a FACS Canto cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by Flowing Software 2 Turku Bioscience Centre, Turku, Finland); the results were expressed as a percentage of positive cells compared to the negative isotype control.

Proteins from EC-EXs and EPC-EXs were isolated with lysis buffer (Thermo Scientific, FL) containing protease inhibitor. Protein concentration assay was conducted using a Bradford assay kit (Bio-Rad Laboratories). The linear range of the assay for BSA is from 0.2 to 0.9 mg/mL. Plates were read at 595 nm using a spectrofluorometer (BioTek Instruments). For western blot analysis, the proteins were subjected to electrophoresis and transferred onto PVDF membranes. The membranes were blocked by incubating with 5% dry milk for 1 hr, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies used were anti-CD63 (1 : 400; BD Biosciences), anti-CD105 (1 : 500; Santa Cruz), anti-CD34 (1 : 500, Santa Cruz), and β-actin (1 : 4000, Sigma). After being washed thoroughly, membranes were incubated with horseradish peroxidase-conjugated IgG (1 : 40000; Jackson ImmunoResearch Labs) for 1 hr at room temperature. Blots were then developed with enhanced chemiluminescence developing solutions.