Ter119

Bone marrow cells were ACK treated and then stained with antibodies Ter119 (BioLegend, 116204, 1/100), B220 (BioLegend, 103204, 1/100), Mac1 (BioLegend, 101204, 1/100), CD3 (BioLegend, 100244, 1/100), Gr1 (BioLegend, 108404, 1/100) (Lin), cKit, Sca1, CD34, Flk2, CD150, IL-7Rα FcγRα (eBioscience, 45-0161-82, 1/100), and PI (Supplementary Table 2). Biotinylated antibodies were stained using Streptavidin-Pacific Orange (Thermo Fisher Scientific) before staining PI. Posttransplant marrow was stained with antibodies Ter119, B220, Mac1, CD3, Gr1, IL-7Rα (Lin), cKit, Sca1, CD34, Flk2, CD150, FcγRα, CD45.1 (BioLegend, 110728, 1/100), CD45.2, and PI.

For cell sorting experiments using mouse HSPCs, cKit⁺ cells were enriched before flow cytometry using cKit magnetic beads (Miltenyi Biotec). Subsequently, the cells were stained with a lineage cocktail, cKit (eBioscience, 17-1171-82, 1:200) and Sca-1 (eBioscience, 25-5981-82, 1:200) antibodies. The lineage cocktail include Gr-1 (Biolegend, 108424, 1:400), Mac-1 (Biolegend, 101226, 1:400), B220 (Biolegend, 103224, 1:400), CD4 (Biolegend, 100414, 1:400), CD8 (Biolegend, 100714, 1:400), CD3 (Biolegend, 100330, 1:400) and Ter-119 (Biolegend, 116223, 1:400). DAPI (1 mg/mL, Sigma-Aldrich) was used to exclude dead cells. For LSC analysis, nucleated BM cells were stained with lineage-specific antibodies, Sca-1 and cKit antibodies. The lineage-specific antibodies include CD3, CD4, CD8, B220, Gr-1, Ter119 and CD127 (Biolegend, 135040, 1:400). For apoptotic analysis, the cells were stained with Annexin V and 7-AAD according to the manufacturer’s recommendations (BD Biosciences, 550475, 1:100). A modified LSR II flow cytometer with four lasers (355 nm, 488 nm, 561 nm, and 633 nm) was used for analyzing, and an Aria III flow cytometer with four lasers (375, 488, 561, and 633 nm) was used for sorting. The analyses were performed using FACSDiVa and the FlowJo (Tree Star) software.

Flow cytometry was performed using an LSRFortessa^TM (Becton Dickinson, Franklin Lakes, NJ, USA) cytometer and FlowJo 10.4 was used to analyze the data.
Antibodies used were: LIFRalpha (FAB5990P; R&D Systems, Minneapolis, MN, USA) and pSTAT3 (557815, Becton Dickinson). Cells were sorted on an Aria III^TM (Becton-Dickinson) as described before [82 (link),83 (link)]. Antibodies used for cell sorting were CD45 (25-0451-82; Thermo Fisher), Ter-119 (116222; Biolegend, San Diego, CA, USA), Gr-1 (108416, Biolegend), CD3 (25-0031-82, Thermo Fisher) and CD11b (25-0112-82, Thermo Fisher). CD31 (102422, Biolegend) Sca-1 (108126, Biolegend), CD166 (12166182, eBioscience, San Diego, CA, USA) and CD140 (135906, Biolegend).