Buffy coats were provided by the blood bank (Sanquin, Amsterdam). Tonsils were obtained from routine tonsillectomies and tissue collection was done at the Academic Medical Center (AMC), Onze Lieve Vrouwe Gasthuis (OLVG) hospital (Amsterdam, The Netherlands). Inflamed nasal polyps were obtained from chronic rhinosinusitis patients during surgery and umbilical cord blood was collected at the AMC. The collection and use of all human samples was approved by the Medical Ethical Committee of the AMC and with informed consent. Peripheral blood and umbilical cord blood mononuclear cells were isolated by Ficoll-Hypaque density gradient (Lymphoprep; Axis-Shield). Tonsil tissue was cut in fine pieces and mechanically disrupted using the Stomacher 80 Biomaster. Cell suspensions were filtered through a 70- µm cell strainer. Nasal tissues were manually cut into fine pieces and digested for 45 min at 37°C with Liberase TM (125 µg/mL) and DNase I (50 U/mL). Cell suspensions were filtered through a 70 µm cell strainer. Mononuclear cells were then isolated by Ficoll-Hypaque density gradient. Subsequently, ILCs were isolated as described previously .
Label
biotin
Label protocol
Briefly, PB and cord blood was enriched for ILCs by immunomagnetic cell sorting, using a negative selection of CD3, CD14, CD16 and CD19 with Mojosort magnetic cell separation system (BioLegend, San Diego, CA, USA) Tonsillar mononuclear cells were depleted of CD3 and CD19 cells by MACS depletion. The cells suspensions were stained with antibodies against Lineage (CD1a, CD3, CD4, CD5, CD14, CD19, CD16, CD34, CD94, CD123, BDCA2, TCRαβ, TCRγδ, FcER1α), CD45, CD161, CD127, CD117, CRTH2, KLRG1, NKp44, NKp46, CD56 and IL-1R1. Cells were sorted on a FACSAria, purity of the sorted cells in all experiments was >99%.
