Milliplex magnetic bead kit

For tumor sample preparation, 5 mg of each frozen tumor was submerged in 0.5 ml of PBS with the recommended concentration of complete Mini Protease Inhibitor Cocktail (11836153001, Roche) and then homogenized using a Bio-Gen™ PRO200® homogenizer for 30 s and centrifuged at 12000 g for 10 min at 4 °C to remove insoluble materials. The supernatants containing the tumor extracts were then quantified using the Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific). Chemokine concentration was measured using the MILLIPLEX® Magnetic Bead Kit (MCYTOMAG-10K, Merck) and following the manufacturer's instructions. For tumor analysis, 10 μg of protein was analyzed, and PBS with protease inhibitor was used as a matrix. For serum analysis, 12.5 μL of serum diluted in assay buffer was added and serum matrix was used. The plate was read on a Luminex® 200™ instrument with xPONENT® software (Merck).

Heparinized blood (10 mL) was obtained from all HMD-AR and mugwort-AR patients, and PBMCs were collected from the blood by Ficoll-Plaque Plus density gradient centrifugation. The PBMCs were stained with the FITC-lineage cocktail as described above and were separated into 2 fractions of Lineage⁺ and Lineage^- cells based on the lineage markers, using a fluorescence-activated cell sorter (BD FACSAriaII; BD Biosciences). The Lineage⁺ and Lineage^- cells were washed by suspension and centrifugation at 300 g for 8 minutes in fresh RPMI 1640 medium, and were then resuspended in RPMI 1640 media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/mL). PBMCs (1.5-2×10⁶ cells/mL), Lineage⁺ cells, and Lineage^- cells (2-2.5×10⁵ cells/mL) were cultured in 96-well round-bottom plates at 37℃ with 5% CO₂ in air atmosphere for 24 hours in the presence of 50 ng/mL IL-25, 50 ng/mL IL-33, or their combination (all from R&D Systems Inc., Minneapolis, MN, USA), together with IL-2 (20 U/mL), a cytokine necessary for lymphoid cell activation.16 (link) At the end of culture, the cell-free supernatants were collected from each well and assessed for the presence of IL-5 and IL-13, using a Milliplex magnetic bead kit (EMD Millipore Corp, Billerica, MA, USA) as recommended by the manufacturer.

BCA-1 (CXCL13), CCL17, CTACK (CCL27), sCD40L, EGF, ENA-78 (CXCL5), Eotaxin (CCL11), FGF, Flt3L, Fraktalkine (CX3CL1), G-CSF, GM-CSF, GRO, I309, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-16, IL-17A, IL17F, IL-20, IL-21, IL-22, IL-23, IL-27, IL28A, IL-33, IP-10, MCP-1 (CCL2), MCP-3 (CCL7), MDC (CCL22), MIP-1α (CCL3), MIP-1β (CCL4), PDGF-AA, PDGF-AB/BB, RANTES (CCL5), SCF, SDF-1, TGF-α, TNF-α, TNF-β, TPO, TRAIL, TSLP and VEGF were measured in the discovery cohort using the Milliplex magnetic bead kit (Merck, USA) as described previously [13 (link)].