Fortessa lsr x20

Cells were treated with drug(s) for 72 hours in a 96-well flat-bottomed, clear microplate. Cell growth and viability was determined by the Alamar Blue assay as per the manufacturers instructions or the sulforhodamine-B (SRB) assay as previously described [44 (link)]. Cell viability and apoptosis was measured by staining cells with Annexin V-FITC (BioVision, Milpitas, CA) and propidium iodide-PE (Sigma-Aldrich, St. Louis, MO) as per manufacturer's instructions. All flow cytometry experiments were carried out using Canto II 96w or Fortessa LSR X20 cytometers (BD Biosciences, San Jose, CA). Flow cytometry data were analyzed with FlowJo version 7.6.5 (TreeStar, Ashland, OR).

Immunoglobulin E (IgE) specific antibodies against 4 allergens (dust mites, mixed moulds, mixed grasses, and mixed animal epithelial) were measured in plasma using an ImmunoCAP Fluorenzyme assay (Pathology North, Newcastle, NSW Australia).
PBMCs were isolated from whole blood by density gradient method (11 (link)) using Ficoll-PaqueTM PLUS (GE Healthcare, Sydney, Australia) and cultured with and without RV1B, LPS or HDM for 48 hours. The concentrations of IFN-γ, IL-1β and IL-6 in the culture supernatants were analysed using bead-based multiplex assay (BD Bioscience, Sydney, Australia) and IFN-λ and IL-5 concentrations in the culture supernatants were measured using high-sensitivity commercial ELISA assays (R&D Systems, Sydney, Australia) as per the manufacturer’s recommendations.
Quantification of major immune cell subsets in whole blood, including eosinophils and neutrophils, T lymphocytes, DCs, NK cells plus B cells and ILCs was performed by multi-parametric flow cytometry using the Fortessa LSR-X20 (BD Biosciences, Sydney, Australia).
For full laboratory analysis methods see supplement.