Sphero rainbow beads

Isolated cells were centrifuged, and then stained for 20 min at 4°C in the dark with various mixes of antibodies (listed in Supplementary Table 1) in brilliant stain buffer (BD Biosciences), after a wash in PBS, we stained the cells with a viability marker [LIVE/DEAD Aqua (Life Technologies)] for 20 min at 4°C in the dark. For intracellular staining, we used BD Biosciences Cytofix/Cytoperm kit, according to manufacturer's instructions. Briefly, after the extracellular staining, cells were permeabilized in Fixation/Permeabilization solution for 20 min at 4°C, cells were then washed twice in Permwash buffer before intracellular staining during 20 min at 4°C. Appropriate isotype antibodies were used as controls. The entire tube of cells was then acquired on a FACS LSR2 (Becton Dickinson). To assess the absolute cell number we used True-count beads (BD Bioscience). Application settings and sphero rainbow beads (BD Biosciences) were used to ensure reproducible and comparable results between patients and over time. BD DIVA software was used for data acquisition and FlowJo (Treestar) software was used for the analysis.

Allergic lung inflammation was induced in Myce and wildtype mice using intraperitoneal ovalbumin (OVA)/alum sensitization followed by nebulized OVA challenge as described previously.
²² (link) On the day after the final challenge, ~200 μL of blood was collected into non‐heparinized tubes by retro‐orbital bleeding, and the mice were then killed by CO₂ inhalation after which BAL was performed (250 μL twice with sterile PBS). Flow cytometry was performed fresh on BAL cells using the following staining panel: CD19‐BUV395 Clone#1D3, CD8a‐PerCPeFluor710 Clone#52–6.7, SiglecF‐PE Clone#E50‐2440 (BD), Ly6c‐eFluor450 Clone#HK1.4, CD11c‐FITC Clone #N418, GR1‐PECy7 Clone#RB6‐8C5, TCRβ‐APCeFluor780 Clone #H57‐597 (eBioscience), CD4‐Alexa647 Clone#GK1.5, CD11b‐Alexa700 Clone#M1/70 from WEHI Antibody Facility (Supplementary table 4). Flow Cytometry was performed on a BD LSRFortessa X‐20 analyzer (BD). SYTOX Blue Dead Cell Stain was used to exclude dead cells from analysis and SPHERO Rainbow Beads (BD) were used to calculate absolute cell counts.