Ms2400 imager

Cytokine analysis was performed on 8 maternal Caspr2 and Control immunized serum pulled to 4 samples each, using a custom U-PLEX Biomarker kit (Meso Scale Discovery (MSD), Rockville, MD), including U-PLEX Mouse IFN-γ, U-PLEX Mouse IL-6, U-PLEX Mouse IL-17A, U-PLEX Mouse IL-23 and U-PLEX Mouse TNF-α. MSD plates were analyzed on the MS2400 imager (MSD). The assay was performed according to the manufacturer’s instructions. All standards and samples were measured in duplicate.

Fecal pellets were collected on select days in PBST (0.1%) and stored until assay. Samples were diluted 1:100 and ELISA was conducted according to the manufacturer’s recommendation (R&D Systems). Optical density was measured using a plate reader, and sample concentrations were calculated by regression analysis.
Total colon homogenates were collected on select days and samples were assayed using V-PLEX Plus Proinflammatory Panel 1 Mouse Kit and V-PLEX Mouse IL-22 Kit (Meso Scale Diagnostics, MSD) according to the manufacturer’s instructions. Briefly, calibrator dilutions, controls, buffers, and plates were prepared according to instructions. Diluted samples with equal protein concentrations were added to each well and left at room temperature with shaking for 2 h. The plates were washed three times and detection antibody solution was added and incubated at room temperature with shaking for 2 h. Read buffer was added to the plates and analytes of interest were measured using MS2400 imager (MSD), and concentrations were extrapolated based on the standard curves.

For the plasma cytokine detection studies, the Human ProInflammatory 7 Ultra-Sensitive Kit from Meso Scale Diagnostics (MSD), which measures IFN-γ, IL-6, IL-8, IL-10, TNF-α, IL-12p70, and IL-1β, was used. MSD plates were analyzed on MSD’s MS2400 imager according to the manufacturer’s instructions. All standards and samples were measured in duplicate.

Media was collected from the luminal (apical) and basolateral sides of the microsnapwell system at 120 min and cytokine levels were quantified using the Human ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso Scale Discovery (MSD), Rockville, MD, US) which measures interleukin (IL)-12p70, IL-1β, interferon (IFN)-γ, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α via multiplex electrochemiluminescence detection. MSD plates were analyzed on the MSD MS2400 imager. All standards and samples were measured in duplicate and the assays were performed according to the manufacturer’s instructions. Cytokine levels were calculated using the manufacturer’s software, given in pg/mL and presented as medians with interquartile ranges. Per the manufacturer’s instructions, the lower limit of detection, in pg/mL, for the assay is 0.77 for IL-12p70, 0.58 for IL-1β, 0.8 for IFN-γ,0.18 for IL-6, 0.10 for IL-8, 0.57 for IL-10, and 0.28 for TNF-α.

The MSD human Proinflammatory-4 II Ultra-Sensitive kit (Meso Scale Discovery) was used to quantify the production of IL-6 and IL-8 following manufacturer’s instruction. Each sample was tested in triplicates in every experiment. MSD plates were analyzed by MS2400 imager from MSD. Duplicate of standards were included in each plate to generate standard curve for data interpretation.