Cd117 fitc

Cells were sorted from 2-3 pooled donors for each replicate as described (Annunziato et al., 2007 (link); Cella et al., 2010 (link); Fuchs et al., 2013 (link)). RNA was isolated (RNeasy Plus Micro Kit, Qiagen), amplified, and hybridized to the Affymetrix Human Gene 1.0 ST arrays. RNA yields from each subset were comparable. Array data were analyzed as described (Robinette et al., 2015 (link)). Unsupervised clustering was performed in R using the hclust function. For CD300LF analyses, CD56–enriched tonsil cells were stained with a PercP-Cy5.5 lineage cocktail containing anti-CD3 (eBioscience), -CD19 (eBioscience), -CD34 (Biolegend) and -ILT3 (Biolegend). Cells were stained with the following combination of antibodies (Biolegend unless indicated): CD117-FITC; NKp44-PE (BD-Pharmingen); CD300f-eFluor660 (eBioscience); CD196-BV421; CD103-BV605; CD161-BV510; CD56-PE-Cy7 (BD-Pharmingen); CD127-biotin (eBioscience), followed by Streptavidin-APC-eFluor780. Data were acquired on an LSR-Fortessa (BD) and analyzed by FlowJo software (TreeStar).

Bone marrow stem cells were extracted from femurs of 6–8-week-old C57BL6 mice and cultured in Dulbecco’s modified Eagle’s medium Glutamax medium, supplemented with 10% FBS, 50 IU penicillin, 50 ug/ml streptomycin and IL-3 for 6 weeks as described.^{49 (link)} IL-3-enriched medium was sourced from murine WEHI-3 myelomonocytic leukaemia cultures and added to growth medium at 20%v/v.^{50 (link)} MC differentiation was verified by cell surface staining with CD117-FITC (fluorescein isothiocyanate; 1:300) and FcεR1α-PE (1:300; BD Biosciences, San Jose, CA, USA). Stained cells were analysed on a LSR-II flow cytometer using the FacsDiva software (BD Biosciences).