Ckit apc cy7

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).

Lin⁻ bone marrow cells were isolated from GFP⁺ or wild-type C57B/6J mice using a Lineage cell depletion kit (Miltenyi). Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells were subsequently purified by FACS sorting on BD AriaIII SORP using PE conjugated antibodies against Lineage⁺ markers (CD3e (clone:145-2C11); CD4 (clone: RM4-5); CD8 (clone: 53-6.7); B220 (clone: RA3-6B2); Ter119 (clone: TER-119); Gr1 (clone: RB6-8C5); Mac1 (clone: M1/70)), Sca1 PE-Cy7 (clone: D7), c-Kit APC-Cy7 (clone: 2B8) (all from eBiosciences).