Pe conjugated anti sca 1

C57BL/6 J (CD45.2) and B6.SJL-PtprcaPepcb/BoyJ mice (B6.SJL, CD45.1) were provided by the Animal Centre of the Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC. Sex- and age-matched mice were used and maintained in the SPF-certified animal facility in the same center. The procedures for the animal experiments were approved by the Animal Care and Use Committee at the institutions involved in this study. After transplantation, the mice were raised in sterile squirrel cages in a positive pressure room. All of the antibodies used in this paper were obtained from eBioscience (San Diego, CA, USA), including PE-, FITC-, or PerCP-Cy5.5-conjugated anti-CD45.1, PE-conjugated anti-CD45.2, PE-conjugated anti-Sca-1, APC-conjugated anti-c-Kit, PE-conjugated anti-CD3, PE-Cy7-conjugated anti-B220, APC-conjugated anti-Mac-1, biotin-conjugated lineage markers (anti-CD3, −CD4, −CD8, −B220, −CD11b, −Gr-1, and -Ter119) and streptavidin-PE-Cy7. Neutralizing antibodies against MIP-3β and CCR7 were purchased from R&D Systems (Minneapolis, MN, USA).

Six male C57BL/6 mice each from the control and cinnamtannin B-1-treated groups were injected with 0.4 mL phosphate-buffered saline (PBS) or cinnamtannin B-1 solution (2.0 mg/kg via the vena caudalis), respectively. The experiments were performed five times. After 1 h, approximately 1 mL samples of peripheral blood were collected, and nucleated cells were separated using Ficoll gradient centrifugation (final density, 1.077 g/m). To detect the endogenous MSCs with the lineage-negative, PDGFRα+, and Sca-1+ (Lin-/PDGFRα+/Sca-1+) cells were stained with allophycocyanin (APC)-conjugated anti-PDGFRα (eBioscience, San Diego, CA, USA), PE-conjugated anti-Sca-1 (eBioscience), or V450 Mouse Lineage Antibody Cocktail (BD Biosciences, San Jose, USA) diluted in PBS and fixed with 1% paraformaldehyde. Flow cytometry analysis was performed using a FACSCanto II flow cytometer (BD Biosciences) using FACSDiva software. The percentage of peripheral blood mononuclear fraction cells that targeted the Lin-/PDGFRα+/Sca-1+ in the control and cinnamtannin B-1 groups was compared.