Immunophenotyping and Signaling Analysis

BM and splenic cells were prepared and analyzed as described previously (Guitart et al., 2017 (link); Guitart et al., 2013 (link); Kranc et al., 2009 (link); Mortensen et al., 2011 (link); Paris et al., 2019 (link); Vukovic et al., 2016 (link)) followed by c-Kit enrichment (described below). Cells were incubated with Fc block and stained with biotin-conjugated anti-Lineage marker antibodies (anti-CD4, anti-CD5, anti-CD8a, anti-CD11b, anti-B220, anti-Gr-1, and anti-Ter119), APC-Cy7–conjugated anti-c-Kit, and Pacific Blue–conjugated anti-Sca-1. Biotin-conjugated Lineage markers were then stained with PerCP-conjugated Streptavidin. Cells were then fixed and permeabilized using Phosflow Lyse/Fix, Phosflow Perm Buffer III, and stain buffer (all from BD Biosciences) according to the manufacturer’s instructions. After processing, cells were stained with AF647-conjugated anti-pStat1 or anti-pStat3.