Isotype matched controls

BM samples were collected from the femurs and tibias of mice and then lysed with erythrocyte lysing buffer. The remaining cells were first stained with Fixable Viability Stain 510 (FVS510; BD Bioscience). For blood cell lineage detection, the cells were stained with lineage antibodies against CD3e, CD11b, Ter-119, B220, Gr-1 and matched isotype controls (eBioscience). To examine the percentages of LSK (Lin^–Sca-1⁺c-Kit⁺) cells, BM cells were stained with biotin-labelled lineage antibodies and then incubated with streptavidin APC-eFluor® 780-labelled secondary antibody (eBioscience), along with BV605-labelled anti-mouse Sca-1 (BD Bioscience) and APC-labelled c-Kit (eBioscience). For the detection of engraftment and chimerism, the BM cells were stained with PE or FITC-labelled antibodies against CD45.1 or CD45.2 (eBioscience). All samples were detected by BD FACS Aria (BD Bioscience).

BM cells were flushed from the femurs of mice with PBS containing 2% FBS. BM RBCs were lysed with lysing buffer (BD). For blood cell lineage detection, the cells were stained with lineage antibodies against CD3, CD11b, Ter-119, B220, Ly-6G and matched isotype controls (eBioscience). To examine the percentages of LSK cells, bone marrow cells were suspended in PBS and incubated with APC-Cy7-labeled lineage antibodies, PerCP-Cy5.5-conjugated anti-Sca-1 and APC-conjugated anti-c-Kit antibodies for 30 min. For the detection of engraftment and chimerism, the bone marrow cells were stained with PE or FITC-labeled antibodies against CD45.1 or CD45.2 (eBioscience). To monitor the proliferating hematopoietic cells, the mice were intraperitoneally injected with BrdU (100 mg/kg body weight) 12 h before sacrifice. The BrdU incorporation assay was performed using a cell proliferation assay kit (Sigma). Cell apoptosis was measured using a PI and Annexin-V staining kit according to the instructions of the manufacturer.

LBC-cell staining was performed as previously described (24 (link)). The following antibodies were employed: anti-mouse CD-24-PE, anti-CD8-PE MAbs, and isotype-matched controls (eBioscience, CA, USA). After incubation with anti-MHC I-Biotin primary antibody, the cells were washed and then incubated with streptavidin–phycoerythrin (PE) conjugated (eBioscience, CA, USA). Fluorescence was measured in a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed with the Flowing 2.5.1 software.