Primary cxcr4 antibody

Lyophilized FITC-labeled E5 and unlabeled E5 were purchased from GL Biochem Ltd. (Shanghai, China). PE mouse anti-human CXCR4 and PE mouse IgG2a (κ Isotype control) antibodies were purchased from BD Biosciences (San Jose, CA). CXCR4 primary antibody was obtained from Abcam (Cambridge, MA). The primary antibodies of Erk, phosphorylated-Erk, Akt, phosphorylated-Akt, p38, phosphorylated-p38 and GAPDH were purchased from Cell Signaling Technology Inc. (Beverly, MA). Protease inhibitor and phosphatase inhibitor cocktail 3 were purchased from Sigma-Aldrich Co. (Germany). Cell lysis buffer was purchased from Cell Signaling Technology Inc. (Beverly, MA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-PE), CXCL12 (SDF-1), and AMD3100 (plerixafor) were purchased from Avanti Polar Lipids (Alabama, USA), R&D Systems (Minneapolis, MN) and Selleckchem (Houston, USA), respectively. Doxorubicin hydrochloride (Dox) was a kind gift from Prof. Wei Liang in Institute of Biophysics. All other chemicals were of analytical grade and used without any further purification.

Flow cytometry (FACS Diva Version 6.1.3 US Becton Dickinson Company) was used to detect BMSC surface markers CD34−CD29 + and CXCR4 +, and the amount of BMSCs in peripheral blood. The PE/Cy5-labeled anti-mouse CD29 (Biolegend, CA, U.S.), PE-labeled mouse monoclonal CD34 (Abcam, U.K.), rabbit polyclonal CXCR4 primary antibody (Abcam), and goat polyclonal anti-rabbit Alexa Fluor^® 488 secondary antibody (Abcam) were used for flow cytometry analyses. The antibodies were added according to manufacturer’s instructions after collection of peripheral blood. Antibodies of the same species and isotype were used for experiments and those that were unrelated were used as negative controls. Cells were incubated for 30 min at 4°C in the dark, washed with cell staining buffer, and fixed in 2% paraformaldehyde at 4°C 30 min before flow cytometric identification of cell surface markers.