Bio plex pro mouse cytokine assay

To detect multiple cytokines in BALF, the Bio-Plex Pro mouse cytokine assay (23-Plex Group I; Bio-Rad) was performed using a Luminex-xMAP/Bio-Plex 200 System with the Bio-Plex Manager 6.2 software (Bio-Rad). Cytokine levels were measured using a cytometric magnetic bead-based assay according to the manufacturer’s instructions.

Concentrations of TNF-α, IL-1β, IL-6 and monocyte chemoattractant protein 1 (MCP-1), were determined in 15 μL of plasma and small intestine PBS homogenates using a multiplex immunoassay kit (Bio-Plex Pro™ Mouse Cytokine Assay, Bio-Rad, Waltham, MA, USA) and measured using Luminex^® technology (Bioplex^®, Bio-Rad, Waltham, MA, USA). The extent of liver inflammation was determined by measurement of plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity using Alanine Transaminase and Aspartate Transaminase Enzymatic Assay Kits (BIOO Scientific Corp., Austin, TX, USA) following the manufacturer’s protocols.

Six cytokines, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6, in sera of immunized mice collected at 3, 7, 14 and 35 DAI were detected using Bio-Plex Pro^™ mouse cytokine assay (Bio-Rad, USA). The levels of IFN-γ⁺ secreting lymphocytes were detected by Mouse IFN gamma ELISPOT Kit (affymetrix eBioscience, USA). Spleen lymphocytes of immunized mice were harvested at 35 DAI and plated (100,000 cells per well) on the ELISPOT plate. The lymphocytes were stimulated with rHSP70 (5 μg/well), rEF-Tu (5 μg/well), Mo extracts (5 μg/well), PHA (0.5 μg/well) or PBS, respectively and incubated at 37°C with 5% CO₂ humidified incubator for 48 hours. Finally, spots were counted using the automated ELISPOT plate reader.

Brain and lung tissues were homogenized as mentioned above and stored at −80°C before further analysis. Prior to detection, the samples were thawed on ice, clarified by centrifugation at 4°C for 10 min at 10,000 rpm/min, and the supernatant was collected. The protein concentration in the supernatants was measured using the Pierce BCA Protein Assay kit [Thermo Fisher Scientific (China) Co., Ltd.] according to the manufacturer’s protocol. All samples were adjusted to the same concentration. The concentration of cytokines and chemokines in the supernatants of tissue homogenates was measured by the Bio-Plex Pro-Mouse Cytokine assay using the Bio-Plex 200 Multiplex Testing System (Bio-Rad, United States) according to the manufacturer’s protocol. The data were analyzed using Bio-Plex Manager software (version 5.0; Bio-Rad, Labs).

Approximately 3 × 10⁵ splenocytes were plated in 96-well plates and stimulated with 1.2 × 10⁴ FFU ZIKV FSS13025 or 10 mg/mL E peptide^{19 (link)} for 3 days. Culture supernatants were harvested and frozen at −80 °C. Cytokines IL-2 and IFN-γ in the culture supernatants were measured using a Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad) according to the manufacturer’s instructions.

IL-1β, IL-6, IL-10, IL-17A, IFNγ and TNFα quantifications were performed on sera from vehicle and CLI-095-treated NOD/BDC2.5 mice with Bio-Plex Pro Mouse Cytokine assay (Bio-Rad, Hercules, USA) according to manufacturer’s instructions.