Cd45 fitc

ADSCs were characterized by flow cytometry at passage 4. Briefly, ADSCs were harvested and washed twice with phosphate-buffered saline (PBS). Then, the cells were incubated for 30 min in PBS containing anti-CD29-FITC (cat. # 555005; BD, San Diego, CA, USA), CD31-PE (cat. # 555027; BD, San Diego, CA, USA), CD49-FITC (cat. # 557457; BD, San Diego, CA, USA), CD90-PE (cat. # 551401; BD, San Diego, CA, USA), CD106-PE (cat. # 559229; BD, San Diego, CA, USA), CD34-FITC (cat. # sc-7324; Santa Cruz, Dallas, TX, USA), CD45-FITC (cat. # MCA43FT; AbD, Oxford, UK), CD73-FITC (cat. # bs-23233R; Bioss, Beijing, China), CD105-FITC (cat. # bs-10662R; Bioss, Beijing, China). The stained cells were then subjected to flow cytometry analysis.

SF and BP were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. GIBCO^® Dulbecco’s modified Eagle medium/F12 (DMEM/F12) was product of Life Technologies (USA). Primary antibodies for CD90-PE, CD45-FITC, CD34-PerCP were from Santa Cruz (California, USA). 2,3,5-triphenyltetrazolium chloride (TTC), MTT and Simvastatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies of VEGF, von Willebrand factor (vWF), brain derived neurotrophic factor (BDNF) and neuronal class III β-tubulin (Tuj1) were products of Abcam (Cambridge, UK). Avidin–biotin peroxidase complex (ABC) kit and 3,3′-diamino-benzidine (DAB) kit were from Zymed Laboratories Inc (California, USA). Primary antibodies of AKT, p-Akt, mTOR and p-mTOR were obtained from Cell Signaling Technology, Inc. (Danvers, USA). The other materials and reagents were from commercial sources.

NPSCs were trypsinised, collected and washed twice with phosphate-buffered saline (PBS). Then, the NPSCs were stained with the following conjugated antibodies: CD29-PE (Biolegend), CD90-FITC (Biolegend), CD44-FITC (Biolegend), CD45-FITC (Santa Cruz) and CD34-FITC (Santa Cruz). An isotype control antibody (Biolegend) was used for each examined antibody. The final antibody concentration was 1 mg/100 mL. After incubating in the dark for 30 min at 37 °C, the cells were washed three times with PBS. Samples were subjected to flow cytometry (Beckman, USA), and the percentage of positive staining was calculated relative to the isotype control staining.