Wallac 1420 manager software

Interleukin (IL)–1β, IL-5, IL-6, IL-10, IL-13, IL-33, tumor necrosis factor alpha (TNFα) and C-C motif chemokine ligand 2 (CCL2) concentrations were evaluated using the Ready-SET-Go! ELISA kits (Thermo Fisher Scientific). CCL24 concentrations were evaluated using the Mouse CCL24/Eotaxin-2/MPIF-2 DuoSet ELISA kit (R&D Systems). Serum Ym1-specific immunoglobulin M (IgM) and OVA-specific immunoglobulin G1 (IgG1) antibody levels were determined in flat-bottom 96-well plates (Greiner Bio-One), coated overnight with 0.1 mg/ml soluble Ym1 in PBS or with 0.1 mg/ml OVA (Worthington) in 0.1 M sodium carbonate buffer (pH 9.5), respectively. After washing and blocking for 1 hr, the samples diluted in PBS were added in duplicate and incubated for 2.5 hr at RT. After washing, biotinylated anti-mouse IgG1 or IgM detecting antibody (0.5 mg/ml; BD Biosciences) plus streptavidin-HRP reagent (1:250; BD Biosciences) was added and incubated for 1 hr at RT. After another wash step, 1×TMB substrate solution (Thermo Fisher Scientific) was added and to stop the reaction, 2.5 N H₂SO₄ was used. Finally, the absorbance was read at 450 nm (and 650 nm as reference) with a Perkin Elmer multilabel counter and data were collected with Wallac 1420 Manager software (PerkinElmer).

Venoms and peptides were plated out into clear curve-based 96-well plates at 5 μg/mL in carbonate buffer and incubated at 4 °C for 24 h. The liquid was removed, and the plates washed three times in a solution of 0.05% TWEEN-20 (“TWEEN-20,” Sigma-Aldrich) in PBS. The primary antibodies were added to the wells with 1:2 dilutions starting from 10 μg/mL in diluent (0.1% bovine serum albumin (BSA) in PBS), and incubated for 1 h at room temperature. The primary antibodies were removed, and the plates washed three times in 0.05% TWEEN-20 in PBS. The polyclonal goat anti-mouse IgG γ-chain-specific secondary antibody was added to the wells (1:1000 in diluent) and incubated for 1 h at room temperature. The primary antibodies were removed, and the plates washed three times in 0.05% TWEEN-20 in PBS. ELISA-developing buffer, a solution of purified water containing 10% citric acid (pH 4.2), 2% ABTS, and 0.1% H₂O₂, was added to the wells, and plates were incubated in the dark at room temperature for 15 min. Absorbance was recorded at 405 nm using the VICTOR Light plate reader with Wallac 1420 Manager Software (PerkinElmer). The control was the mouse monoclonal IgG antibody (28/00 8C1-6) that reacts with human IL-12, applied to the melittin peptide on the ELISA plate. Experiments were conducted in biological replicates (n = 3).