Elisa

Immunity was assessed on day 10 after immunisation with 20μg MPO in FCA and at day 21, at the end of the autoimmune anti-MPO glomerulonephritis model. The spleen and draining lymph nodes were harvested and a single cell suspension was obtained. IFN-γ and IL-17A ELISPOT was performed according to the manufacturer’s instructions (Ebioscience, San Diego, CA) with 5x10⁵ cells per well. Cells were incubated for 18 hours at 37°C with 10μg/ml heat inactivated recombinant murine MPO (rMPO). Total anti-MPO IgG was measured by ELISA on MPO coated plates, with IgG detected with sheep-anti mouse IgG-HRP (Sigma-Aldrich). Antibody subclasses were measured using subclass specific goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL). Serum BAFF was measured by ELISA (RnD Systems, Minneapolis, MN). For assessment of B cell development, bone marrow was flushed from tibiae and femora of naïve mice and analysed by flow cytometry.

Hemodynamic parameters were continuously monitored including MAP, mean pulmonary artery pressure (MPAP), right atrial pressure (RAP), cardiac output (CO), cardiac index (CI), and S_vO₂.
Blood was sequentially drawn for the determination of (i) blood gases, (ii) arterial lactate, (iii) plasma concentration of urea, creatinine, albumin (VetTest GHP, Idexx, Saint-Denis, France) (iv) blood cell count, and (v) TNF-α, and IL-6 (ELISA, RnD Systems, Minneapolis, USA). At the end of the experiment, bacterial counts were performed on blood samples, and lung and kidney biopsies were taken for histological analyses. Histology scores were determined by an experienced pathologist who was blinded to the treatment applied.

HMGB1 levels were determined in plasma or MVs by ELISA (IBL, Germany) according to the manufacturer’s instruction with modification. Plasma samples were diluted 1:25 in Tris lysis buffer containing 7.4% EDTA, 3.8% EGTA and 1% Triton X-100. Samples were then treated with perchloric acid (PCA, BioVision catalog #K808) to liberate HMGB1 from its binding partners to allow for measurement of total HMGB1 as previously described [40 (link)]. MV samples were added directly to ELISA after PCA treatment without prior dilution. The purified HMGB1-containing supernatant was then assessed by ELISA. IL-1β was measured in MVs and plasma by ELISA (RND systems, DY401) according to the manufacturer’s instructions. Protein concentrations of MVs and plasma were assessed using a BCA protein determination kit (Thermo Fischer #23225). HMGB1 and IL-1β measurements in MVs were normalized to total MV protein to account for sample variation. Previously in our laboratory we have observed that there is a Hook effect when measuring HMGB1 in plasma samples, which results in erroneously low plasma HMGB1 measurements when a low dilution factor is used. Therefore, a dilution factor of 30, optimized from our previous work, was used for all plasma HMGB1 ELISA assessments.

BMDCs or splenic DCs (CD11c⁺) were plated at 2.5 × 10⁴ cells per well in a U-bottomed 96 well plate. BMDCs were treated with oligomycin (10 μM), Trifluoromethyoxy carbonlcyanide phenylhydraxone (FCCP; 10 μM) or ruthenium red (10 μM) for 3 h before addition of Ovalbumin (25 μg/mL) for 2 h followed by treatment with LPS (10 ng/mL) for 4 h (or remainder of experiment). Cells were then washed with PBS to remove inhibitors from culture medium and prevent any carry over effects on T cells. CD4+ T cells were isolated from spleens of OTII transgenic mice using CD4 negative selection kit (Stem Cell) according to manufacturer’s instructions. 1.25 × 10⁵ CD4⁺ T cells were then added to the wells containing treated BMDCs or splenic DCs (CD11c+) and incubated for 3 days. Supernatants were collected on day 3 and analysed for IFNγ and IL17 by ELISA (RnD systems).

Supernatants from co-cultures for analysis of chemokine production were harvested after 96 hrs culture, centrifuged at 350 x g to remove cells and debris. Supernatants were then stored at -20°C prior to analysis. A Legendplex^® mix and match bead array assay was used to detect production of CXCL13, CCL22 and CX3CL1 according to the manufacturer’s instructions. CXCL13 production was also measured by ELISA (RnD systems). Active TGF-β was measured by ELISA according to the manufacturer's instructions (Biolegend).

The concentrations of GDNF, BDNF, NGF and CNTF in CM were measured by ELISA (RnD systems, Minneapolis, MN, USA) in accordance with the manufacturer’s protocol. The collected CM samples were concentrated 24-fold with 3 kDa Amicon Ultra filter units (Sigma-Aldrich, St. Louis, MO, USA) to 0.5 mg/mL total protein concentrations prior to the analysis. Optical densities (absorbance at 450 nm) were measured in a plate reader (PerkinElmer, Waltham, MA, USA). For evaluation of the concentrations of neurotrophins, the experiment was repeated at least 3 times.