Uncoated elisa kit

No commercial kits for determining the levels of Der p 1 on EVs are available. Therefore, we developed an ELISA assay for Der p 1 on EVs. The detailed methods are presented in Supplemental Materials. Level of IL-13 in plasma and cell culture supernatants was assayed using high-sensitivity commercial ELISA kit purchased from Neobioscience (Shenzhen, Guangdong, China) and Uncoated ELISA kit purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Media supernatants collected after co-culture were analyzed by ELISA for human IFN-γ levels using the Uncoated ELISA Kit from Thermo Fisher Scientific according to manufacturer’s instructions (Cat. no: 88-7316-88). Each experiment was performed at least three times in technical duplicates.

Plasma levels of IL-6 and TNF-α were quantified from EDTA plasma by ELISA using a commercially available kit (Uncoated ELISA kit, ThermoFisher Scientific) according to the manufacturer’s protocol. Plasma levels of CXCL12 were quantified in the same fashion using a commercially available kit (Quantikine ELISA kit, R&D Systems, San Diego, CA, USA).

Mouse IL-6 was quantified by uncoated ELISA Kit (ThermoFisher) according to manufacturer’s instruction.

HeLa (human epithelial; ECACC no. 93021013), RAW264.7 (murine macrophages; ECACC no. 91062702), and NRK-49F (normal rat kidney fibroblasts; ATCC CRL-1570) cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were kept in a humidified atmosphere with 5% CO₂ at 37°C. For cell lysis, 2 × 10⁷ to 10⁸ cells per ml were incubated at 4°C in NP40 buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP40, 1 mM PMSF, 1% protease inhibitor cocktail P8849 from Sigma-Aldrich) for 20 min. The extract was centrifuged at 20,000× g for 20 min and the supernatant was stored at -80°C. For transient transfection assays, 2–5 × 10⁶ HeLa cells/assay were resuspended in 200 μL of 15 mM HEPES-buffered serum-containing medium, mixed with 50 μL of 210 mM NaCl containing 5–10 μg of plasmid DNA and electroporated using a BTX Electrocell Manipulator 600 set at 240 V, 950 μF, resistance = None. Cells were processed 24 h after electroporation. The supernatants of transfected cells were tested for CCL5 secretion using the human CCL5/RANTES DuoSet ELISA kit (R&D Systems) and for the secretion of IFNγ, IL-1β, IL-6 and IL-8 using the appropriate uncoated ELISA kits (Thermo Fisher Scientific).

Cells were seeded in 6-well plates at density of 30,000 cells per cm² and grown for 24 h before the treatments. All treatments are described in the legends to the Figures. After treatment, the culture medium was aspirated and stored at −20 °C until further analyses. The content of cytokines was measured using Uncoated ELISA kits (Thermo Fisher Scientific), for IL-6, IL-8 and CCL2/MCP1, respectively. Briefly, high-affinity protein binding plates were incubated with capture antibody overnight. The wells were then blocked with PBS supplemented with fetal bovine serum for 1 h. After washing, the plates were incubated with conditioned media for 2 h. Subsequently, wells were incubated with the detection antibody for 1 h. After washing, Streptavidin-HRP was added to all wells and incubated for 30 min. Finally, substrate solution was added, incubated for 15 min and the reaction was stopped. Optical densities were measured at 450 nm.

BALF was collected by injecting 1 mL PBS into the trachea of each mouse, and the solutions were centrifuged at 800 x g for 5 min at 4 °C. Each supernatant was transferred to a fresh tube for cytokine analysis, whereas each cell pellet was used for cell counting and immunotyping. To identify cell populations, the cells were incubated with antibodies against CD11b (clone: M1/70, BioLegend), Gr1 (clone: RB6‐8C5, BioLegend), F4/80 (clone: BM8 BioLegend), and CD45 (clone: 30‐F11, BioLegend) and subjected to flow cytometry (BD Accuri C6). Apoptotic cells were detected using a FITC Annexin V Apoptosis detection kit (catalog no. 556 547, BD Biosciences). Cytokine concentrations in BALF were measured by conventional ELISA using uncoated ELISA Kits (ThermoFisher Scientific) for mouse IL‐1β (catalog no. 88‐7013‐88), IL‐6 (catalog no. 887064–88), IL‐4 (catalog no. BMS613), and IL‐10 (catalog no. 88‐7105‐88).