Pe mouse anti human cd105

The criteria of International Society for Cellular Therapy were used to characterize MSCs [13 (link)]. To evaluate immunophenotypic expression, cultured MSCs were detached, washed, and resuspended in phosphate-buffered saline. After fixing and blocking, cells were immunolabeled with phycoerythrin (PE) mouse anti-human CD34 (Clone 8G12; BD Biosciences, San Jose, CA, USA), fluorescein isothiocyanate (FITC) mouse anti-human CD45 (Clone 2D1; BD Biosciences, San Jose, CA, USA), allophycocyanin (APC) mouse anti-human CD44 (Clone G44-26; BD Biosciences, San Jose, CA, USA), or PE mouse anti-human CD105 (Clone 266; BD Biosciences, San Jose, CA, USA) antibodies. Mouse IgG (BD Biosciences, San Jose, CA, USA) served as isotype control. Data were analyzed by flow cytometry (Accuri C6; BD Biosciences, San Jose, CA, USA). To assess differentiation potential, cultured MSCs were detached and replated in 60-mm dishes. For osteogenic induction, MSCs were grown in DMEM with 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 0.2 mM ascorbic acid. For adipogenic induction, MSCs were grown in DMEM with 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 0.1 mM indomethacin, and 10 μg/ml insulin. After 2-week induction, osteogenesis and adipogenesis were demonstrated by von Kossa stain (Cedarlane, Ontario, Canada) and oil red O stain (Sigma, St Louis, MO, USA), respectively.

Protein expression was evaluated in EA.hy926 cells and MLECs by “Western blot”, “Flow cytometry” and “Cellular immunofluorescence”. Antibodies for western blot: anti-human endoglin (Ref. H300; Thermo Fisher Scientific), anti-mouse β-actin (Ref. A5441; Sigma-Aldrich). Antibodies for flow cytometry: PE mouse anti-human CD105 (Ref. 560839; BD Biosciences). Antibodies for immunofluorescence: anti-human endoglin hybridoma TEA1/58.1 (provided by Dr. Sánchez-Madrid, CNIC, Spain), anti-mouse VE-cadherin (Ref. ab33168; Abcam) and secondary antibody anti-mouse IgG AlexaFluor568^® (Ref. A10037; Thermo Fisher Scientific) were used. The “patching algorithm” for MATLAB was also used for VE-cadherin patterning quantification in EA.hy926 ECs.

The expression levels of SSEA-3 (derived using anti-SSEA-3; 1:300, Abcam, Cambridge, UK, ab16286) and CD-105 (derived using a PE mouse anti-human CD-105; 1:300, BD Pharmingen; catalog no. 560839) were determined via immunofluorescence staining. After 2 days of culture in the medium described above, hNTSCs were fixed in 2% (w/v) paraformaldehyde and washed with phosphate-buffered saline. The cells were then permeabilized with 0.3% (v/v) Triton X-100 (Sigma-Aldrich) and washed with phosphate-buffered saline. After cells had been blocked with 1% (v/v) normal goat serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), they were incubated with the primary antibodies mentioned above; they were then incubated with a goat anti-rat Alexa-Fluor 488 antibody (1:1000; Molecular Probes). The nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and fluorescence was observed under a Zeiss LSM510 confocal microscope (Carl Zeiss).