Ficoll hypaque density gradient lymphoprep

Human pediatrics tonsils were collected with approval of tissue‐specific protocols by the Medical Ethical Committee of the Amsterdam UMC, Amsterdam. Tonsils were obtained from pediatric tonsillectomies performing within the Amsterdam UMC or Onze Lieve Vrouwe Gasthuis hospital (Amsterdam, The Netherlands). Informed consent was obtained from patients whose tonsils were used in this study after the nature and possible consequences were explained. Buffy coats were provided by the blood bank (Sanquin, Amsterdam). PBMCs were isolated by Ficoll‐Hypaque density gradient (Lymphoprep; Axis‐Shield). Tonsils were processed to obtain cell suspensions as described before [40]. For ILC enrichment, tonsil cell suspensions were depleted for CD3 and CD19 by MACS depletion and PBMC were depleted for CD3, CD14, and CD19 using MojoSort magnetic cell separation system (BioLegend, San Diego, CA, USA). Obtained (depleted) cell suspensions were stained for 30 min at 4°C with fluorochrome‐conjugated antibodies (Supporting Information Table S1). The cells were purified by flow cytometry using a FACSAria IIU (BD Biosciences).