Milliplex analyte software

Stimulation tubes were thawed and brought to RT before use. Within 90 min of the blood draw (up to 6 h for CF/ABPA/CPA patients), 500 µL WB was injected into each stimulation tube with a graduated 1 mL insulin syringe. Stimulation tubes were inverted 10 times and incubated for 24–26 h at 37 °C. Plasma was collected after a 20 min centrifugation step at 2000 g and cryopreserved at −20 °C. Cytokine concentrations were determined using IFN-γ and IL-17 ELISA Max Deluxe Sets (Biolegend, San Diego, CA, USA) following the manufacturer’s instructions. Absorbance was read on a NanoQuant Infinite 200M Pro microplate reader (Tecan, Maennedorf, Switzerland) and cytokine concentrations were interpolated from a 7-point standard curve after subtraction of baseline absorbance in a plain medium control. The Milliplex® MAP human high-sensitivity T Cell magnetic bead panel kit (Merck, Darmstadt, Germany) was used for the multiplex cytokine assay. Cytokine concentrations were determined using a Luminex200 reader (Luminex Austin, TX, USA) in combination with the XPONENT3 (Luminex Austin, TX, USA) and Milliplex Analyte software (Merck, Darmstadt, Germany).

Anticoagulant-free stimulation tubes were prepared as detailed in Table S2, following a previously published protocol [12 (link)], and cryopreserved at −20 °C for up to four weeks. Prior to whole-blood (WB) stimulation, the ready-to-use stimulation tubes were brought to room temperature. Within 90 min of blood collection, 500 µL of WB was injected into the tubes with a graduated 1-mL insulin syringe. Stimulation tubes were inverted 10 times and incubated for 24 h at 37 °C. Plasma was collected by centrifugation at 2000× g for 20 min and cryopreserved at −20 °C. Cytokine concentrations were determined using a 21-plex Milliplex^® MAP human high-sensitivity T-cell magnetic bead panel kit (Merck, Darmstadt, Germany) and a Luminex200 reader (Luminex, Austin, TX, USA) in combination with the XPONENT3 (Luminex, Austin, TX, USA) and Milliplex Analyte software (Merck, Darmstadt, Germany). Cytokine concentrations were interpolated from 7-point standard curves.