Gating strategy for ILC2 sorting.

After depletion of CD3-, CD14- and CD19-positive cells from PBMCs via magnetic activated cell separation (MACS), cells were stained with a PerCP-Cy5.5-labeled lineage cocktail 1 (CD4, CD8, CD14, CD16, CD19, CD34, CD123, FcεRI), a FITC-labeled lineage cocktail 2 (CD11b, CD56), CD3-BV510, CD127-BV421, CRTH2-PE and CD45-Alexa Fluor 700. (a) Lymphoid cells were gated by cell size (FSC-A) and cell granularity (SSC-A), and (b) discriminated from doublets (clotted cells) by determination of the time of flight that each cells needed to pass the filter (SSC-W). For further gating, (d, f, g, i, j) gates were set according to FMO controls. (c) CD45^high, lineage1^-, (e) lineage2^-, CD3^-, (h) CD127⁺ and CRTH2⁺ cells were defined as ILC2 and sorted into 96 U buttom well plates containing 100 μL supplemented medium. Gate boundaries were not altered between participants.

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