Annexin 5 apoptosis detection kit 1

ILCs were sorted from fresh PBMCs as CD117⁺CD127⁺CD56⁺ cells, as described elsewhere.^{11 (link)} Separately, CD3⁻CD14⁻CD19⁻CD117⁻CD127⁻ CD56⁺ NK cells and CD3⁺ T cells were also sorted. For comparison with ILCs from patients with CVID and control subjects, ILCs were similarly sorted from splenocytes of fresh spleens from healthy subjects removed because of trauma.^{11 (link)} Cells were stained with appropriate mixtures of fluorochrome-labeled antibodies (see Table E1) and sorted with a FACSAria II (BD Biosciences) after exclusion of dead cells by using the LIVE/DEAD Fixable Violet Cell Stain Kit (Invitrogen, Carlsbad, Calif). The purity of sorted cells was consistently greater than 97%. To further examine sorted circulating ILCs, cells (5 × 10⁴/well) were plated in 96-well U-bottom plates and then cultured for 3 to 5 days in complete RPMI medium with 10% FBS, penicillin, and streptomycin (10 U/mL), with or without 50 ng/mL IL-7, 50 ng/mL IL-1β, or both (PeproTech, Rocky Hills, NJ), as previously described.^{11 (link)} The survival of the sorted ILC population after culture was measured with the Annexin V Apoptosis Detection Kit I (BD PharMingen). Cells were acquired with an LSR Fortessa (BD) and analyzed by using FlowJo software (Tree Star), with comparisons with isotype-matched antibody controls.