Percp cy5 5 conjugated anti cd45

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).

Cells derived from in vitro mouse PSC hematopoietic differentiation and the in vivo PB and BM of m-NSG mice were harvested and suspended in PBS with 2% FBS. Before antibody incubation, the cells were blocked with an anti-CD16/32 antibody (eBioscience). The following antibodies were used for analysis: Alexa Fluor 700 (AF700)-conjugated anti-Flk1 (eBioscience), allophycocyanin (APC)-conjugated anti-TIE2 (eBioscience), APC-Cy7-conjugated anti-c-kit (eBioscience), AF700-conjugated anti-Lineage (eBioscience), APC-conjugated anti-Sca-1 (eBioscience), peridinin-chlorophyll protein (PerCP)-eFluor® 710-conjugated anti-CD201 (eBioscience), APC-Cy7-conjugated anti-TER119 (eBioscience), PerCP-Cy5.5-conjugated anti-CD45.1 (eBioscience), and PE-Cy7-conjugated anti-CD45.2 (eBioscience). The following antibodies were used for sorting: APC-Cy7-conjugated anti-c-kit (eBioscience), eFluor450-conjugated anti-Lineage (eBioscience), and phycoerythrin (PE)-conjugated anti-CD201 (eBioscience). Samples were measured by BD Fortessa, and cells were sorted by BD Aria II. The data were analyzed using FlowJo Version 10 software.