Anti ige

All ELISAs were performed using Costar half-area, high-binding plates. For worm-specific IgE, plates were coated with 20 μg/mL LsAg in PBS and incubated overnight at 4°C. Plates were blocked with PBS/5% BSA and 0.05% Tween 20. IgG was depleted out of plasma using GammaBind plus Sepharose beads (GE Healthcare Biosciences). Plasma was then diluted in 1% BSA/PBS and 5-fold serial dilutions were made. Samples were added to the plate in duplicate. Plates were then washed and plate-bound IgE detected by the addition of biotinylated anti-mouse IgE (BD Biosciences) diluted in 1%BSA/PBS. Following another wash, alkaline phosphatase conjugated streptavidin (Jackson Immuno Research Labs) diluted 1:1000 in 1%BSA/PBS was added. Plates were developed by the addition of 4-nitrophenyl phosphate disodium (4-NPP, Sigma-Alderich) in 0.1M carbonate buffer. Absorbance was read at 405nm using a Victor³ V microplate reader from PerkinElmer. Detection of total IgE was performed with identical steps as for parasite-specific IgE except that plates were initially coated with 10 μg/mL anti-IgE (BD Biosciences)in PBS and plasma samples were plated at dilutions of 1:10 and 1:90. Total IgE concentrations were determined using an IgE standard curve (BD Biosciences) and WorkOut 2.0 ELISA software (PerkinElmer).

Mice were sacrificed at week 78 of the protocol and single-cell suspensions of splenocytes were prepared in ice cold staining buffer (PBS including 0.5 mM EDTA, 0.05 mM sodium azide, 0.5% BSA). First, surface staining with unlabeled anti-IgE (to block membrane IgE), APC anti-CD138, BV711-anti-CD3, and anti-CD16/32 (Fc-block) (all from BD Biosciences), CA was performed. Live-dead discriminating dye (Live-Dead Aqua, Invitrogen, CA) was included. Cells were washed and incubated with fixation/permeabilization buffer (BD Biosciences, CA) for 15 mins, washed with permeabilization buffer (BD Biosciences, CA), and then incubated with FITC-anti-IgE, in permeabilization buffer. After washing, cells were treated with Cytofix buffer (BD Biosciences, CA) for 15 mins for post-fixation, washed, and then data were acquired on an LSRII flow cytometer (Becton Dickinson, CA). Flow cytometry analysis was performed using Flow Jo (Tree Star, CA) as follows. Live singlet cells were then analyzed for IgE⁺ plasma cells (FITC-IgE +; APC-CD138+ cells).

For the Treg/CD11b⁺myeloid cell co-culture assay, 1 × 10⁴ CD45⁺CD11b⁺ cells sorted from the brains and spinal cords of day 10 MOG-immunized mice were co-cultured with 5 × 10³ splenic YFP⁺(FoxP3⁺)CD4⁺CD3⁺ Tregs sorted from these mice in a 96-well round-bottom plate for 40 h followed by FACS analysis of Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) expression in CD11b⁺ cells. IL-33 (30 ng/ml) was added into CD11b⁺ cells or the co-culture groups. Isolation of myeloid/microglia from adult mouse brains was performed according to the published methods [21 (link)]. Briefly, mouse brains were harvested after perfusion and digested for 20 min in the dissociation medium (DMEM/F12 medium supplemented with 1 mg/ml papain (Cat# ICN10092180, Fisher Scientific), 1.2 U/ml dispase II (Cat# NC1136921, Fisher Scientific) and 20 U/ml DNAse I). The cell suspension was collected, filtered through a 40-μm cell strainer, and then separated on a 30-37-70% percoll gradient followed by collecting the 37-70% interphase. 5 × 10⁴ cells per well were seeded in a 24-well plate and cultured in DMEM/F12 medium with 10% FBS overnight. EAE sera were added into the culture for 48 h, followed by FACS analysis of CD11b⁺ cells. Cells treated with anti-IgG (Cat# 115-005-008, Jackson ImmunoResearch) or anti-IgE (Cat# 553416, BD Biosciences) were included as controls.

IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM were measured using a kit (Mouse Isotyping Panel 1 kit) from MSD according to their protocol. Plasma samples were diluted 1∶1000, to 1∶125,000 to ensure samples reading within the linear range of the assay for each antibody.
To measure total IgE, standard 96-well MSD plates were coated (overnight, 4°C) with anti-IgE (2 µg/ml, PBS, BD). All further incubations were carried out at room temperature on an orbital shaker. Unbound antibody was removed by washing (0.05% Tween 20 in PBS), and non-specific binding blocked (1% BSA/PBS, 200 µl/well) for 1 h. The plate was washed, 25 µl standard (14–10,000 ng/ml, purified mouse IgE, BD) or sample (diluted 1∶10) added and incubated for 2 h. Unbound antibody was removed by washing and 25 µl biotinylated anti-mouse IgE (1 µg/ml; BD) pipetted per well. Following 1 h incubation, the plates were washed and 25 µl streptavidin Sulfo-Tag (1 µg/ml; MSD) added for a further 1 h. Plates were then washed, 150 µl Read buffer (T- x2; MSD) added per well and the samples analysed on SI6000 (MSD). OVA-specific IgE was measured by ELISA (AbD Serotec) according to their protocol.

Anti-IgE (0.5mg/mL, clone G7-18, BD Bioscience), f-Met-Leu-Phe (fMLP, 5mg/mL, Sigma-Aldrich), stem cell factor (SCF, Miltenyi Biotec, Bergisch Gladbach, Germany) were used.