B220 apc

Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

For immunophenotypic analysis of lineage positive cells, PB, BM and spleen samples were processed with 1× red blood cell lysis buffer, and then incubated with CD11b-PE-cy7 (25–0112-81, eBioscience), Gr1-eFluor450 (48–5931-82, eBioscience), CD3-PE (12–0031-83, eBioscience) and B220-APC (17–0452-82, eBioscience). To distinguish donor from recipient hematopoietic cells, PB were stained with CD45.1-Brilliant Violet 510 (110741, BioLegend), and CD45.2-APC-eFluor780 (47–0454-82, eBioscience) or CD45.2-eFluor450 (48–0454-82, eBioscience). For HSPC analysis, BM cells were washed and incubated for 30min with biotin conjugated lineage markers (CD11b, Gr1, Ter119, CD3, B220, mouse hematopoietic lineage biotin panel (88–7774-75 eBioscience)), followed by staining with streptavidin eFluor780 (47–4317-82, eBioscience), Sea-1-PE (12–5981-82, eBioscience), c-Kit-APC (17–117181, eBioscience), Flk2-PE-Cy5 (15–1351-81, eBioscience), CD150-PE-Cy7 (115914, BioLegend) and CD48-FITC (11–0481-85, Affymetrix) or CD48-eFluor450 (48–0481-80, eBioscience). SLAM-HSC were identified based on expression of Lin⁻Sca⁻1⁺c-Kit⁺CD150⁺CD48⁻. MPP2, MPP3 and MPP4 were identified based on expression of Lin⁻Sca^_1⁺c-Kit⁺Flk2⁻CD150⁺CD48⁺, Lin⁻Sca⁻1⁺c-Kit⁺Flk2⁻CD150⁻CD48⁺ and Lin⁻Sca⁻1⁺c-Kit⁺Flk2⁺CD150⁻CD48⁺, respectively.