Quickplex sq 120 instrument

The levels of cytokines released into the medium on treatment days 13 and 27 were assayed on the MESO QuickPlex SQ 120 Instrument using the Meso Scale Discovery V-PLEX Human Proinflammatory Panel 1 kit according to the manufacturer’s instructions. Samples and standards were prepared with minor modifications as described previously [9 (link)]. Only cytokines previously shown to be involved in modulating fibrogenic processes were analyzed [19 ]. Cytokine data have been deposited in the Dryad Digital Repository and can be downloaded at doi: 10.5061/dryad.5rc1973.

A customized, high-sensitive 10-cytokine U-plex panel (Meso Scale Discovery, Rockville, MMD, USA) was used to analyze plasma levels of IFNβ, IFNγ, IL10, IL1β, IL21, IL6, IL8, IP10, MCP1, and TNFα in cryopreserved plasma samples according to the manufacturer’s protocol. Data were acquired using a QuickPlex SQ 120 instrument (Meso Scale Discovery, Rockville, MD, USA). The lower limit of quantification of each cytokine can be found in Table S2.

ILC3s were isolated from colonic LPMCs and on average purified to 91.79% purity (Supplemental Figure 2) by sorting using the MoFlo Astrios EQ (Beckman Coulter, Indianapolis, IN). ILC3s were sorted from Viable CD45+ single cell lymphocytes that were Lineage-CD127+CD117+ as described above. Purified ILC3s were then exposed to either 50 ng/mL IL-23 and IL-1β (R & D Systems) or whole bacteria at a ratio of 1 bacteria to 1 ILC3 and incubated for 24 h at 37°C + 5% CO₂. Supernatant was collected and saved at −20°C until use. Secreted IL-22 was measured in the supernatant using the IL-22 U-PLEX Assay according to manufacturer's instructions and quantified on the QuickPlex SQ 120 Instrument (Mesoscale Discovery, Rockville, MD).

Inocula were prepared by mixing DENV with antibody dilutions (ADE conditions) or media (virus-only condition) and incubating at 37°C/5% CO₂ for 60 minutes to allow for immune complex formation. Supernatants from 6-day monocyte-derived macrophage cultures were aspirated and 200 μL of inoculum added to each well. Plates were rocked to ensure even coverage by the inoculum and incubated for 2 hours at 37°C/5% CO₂. After incubation, the inoculum was aspirated, wells were washed twice with 2mL RPMI medium, 500 μL differentiation media added, and plates incubated for 48 hours at 37C/5% CO₂. After 48 hours, the supernatant was removed, aliquoted, and stored at -80C for cytokine expression and viral burden analyses. Macrophages were detached from the plates by adding 600 μL of Accutase (Stemcell technologies, 07920) per well and incubated at 37°C for 20 minutes, dissociated by pipetting, transferred to round-bottom polypropylene 96-well plates, and analyzed as described for each respective assay. Cytokine production from DENV infection macrophages was quantified using an MSD QuickPlex SQ120 instrument and a human U-PLEX cytokine panel (Meso Scale Diagnostics).

Plasma was thawed on ice and TNFα, IL-6, IFNγ, and IL-1β were quantified by the U-PLEX Assay and CRP was quantified using the V-PLEX Assay according to manufacturer’s instructions (Meso Scale Discovery, Rockville, MD). Both assays were evaluated on the QuickPlex SQ 120 Instrument (Meso Scale Discovery).