Mouse il 10 duoset elisa

Cytokines were quantified, according to the manufacturer, using the commercial kits: Mouse IL-10 DuoSet ELISA, (R&D Systems, MN, USA), IL-12p70, IL-4 and IFN-γ ELISA MAX Deluxe (BioLegend, CA, USA).

6 x 10⁵–7 x 10⁵ BMMs in 500 μL BMM growth media were seeded into wells of a 24-well plate. Bacteria were grown overnight at 30°C at a slant without shaking in 3 mL Brain-Heart Infusion Broth containing 200μg/mL streptomycin. Cultures were then pelleted by centrifugation, and resuspended in phosphate-buffered solution (PBS) to an optical density of 2.0. Wells were infected with 20μL of bacteria, approximately 8 x 10⁷ CFU (MOI = 120). Three wells were infected for each bacterial strain. 30 minutes post-infection, cells were washed with warm PBS, and BMM growth media with 50 μg/mL gentamicin was added. Supernatants were collected and frozen at -80°C until used for analysis. For quantification of IL-10, Mouse IL-10 DuoSet ELISA (R&D Systems) was performed according to manufacturer’s instructions. Data was analyzed using GraphPad Prism.

Measurement of the mouse cytokine levels (IFN-γ, TNF-α, IL-6, IL-10, IL-12) was performed with ELISA in conditioned medium from MSCs during 1–13 passages and also in a medium from a selection of the MSCs transfected with pcNS3-NS5B. IFN-γ quantification was also conducted in mice sera after second immunization. We used the Mouse IL-6 ELISA development kit (HRP), Mouse IFN-γ ELISA development kit (HRP), Mouse TNF-α ELISA development kit (HRP) (Mabtech, Stockholm, Sweden), Mouse IL-10 DuoSet ELISA, and Mouse IL-12 p70 Duoset ELISA (R&D Systems, Minneapolis, MN, USA). The detection sensitivity for IL-6 was 10 pg/mL, for IFN-γ and TNF-α 2 pg/mL, and for IL-10 and IL-12 30 pg/mL.
Human cytokine secretion by Huh7.5 cells was assayed by ELISA, using Vektor-Best kits (Russia) for IL-1β, IL-6, IFN-γ, and TNF-α. The sensitivities of the cytokine assays were 0.5 pg/mL for IL-6, 1 pg/mL for IL-1β and TNF-α, and 2 pg/mL for IFN-γ. The concentrations of cytokines were determined from the calibration curves of the standard samples.

Splenocytes from immunized and adjuvant control animals were plated in U-bottom cell culture 96 well plates (Orange Scientific, Braine-l’Alleud, Belgium), and stimulated ex vivo with 10 µg/mL of recombinant PyCSP for 72 h, at 37°C, 5% CO₂. Supernatants were collected and stored at -80°C until use. Cytokines were quantified in the supernatants using commercial kits, according to manufacturer’s instructions: Mouse IL-10 DuoSet ELISA (R&D Systems, Minneapolis, Minn, USA), mouse IL-4, IL-6, IL-2, TNF-α and IFN-γ ELISA MAX (BioLegend).
Sera of immunized and adjuvant-control animals was collected 5 hours post prime immunization and analyzed using the LEGENDplex™ Mouse Anti-Virus Response (13-plex) Panel (BioLegend), according to manufacturer’s instructions using a BD FACSCanto II flow cytometer (BD Biosciences) running BD FACSDiva Software version 6.1.3 (BD Biosciences). Results were analyzed using FlowJo version 10.7.1 (FlowJo LLC) and the concentration of the cytokines IFN-γ, TNF-α, IL12(p70), IL-1β, GM-CSF, IL-10, IFN-β, IFN-α, IL6, and chemokines CXCL1 (KC), CCL2 (MCP-1), CCL5 (RANTES) and CXCL10 (IP-10) determined, in pg/mL.

IFN-γ, IL-10, and IL-12 were quantified by ELISA in the conditioned medium samples obtained in the lymphocyte proliferation assays. The following ELISA systems were used: Mouse IFN-γ ELISA development kit (HRP) (Mabtech, Nacka Strand Sweden), Mouse IL-10 DuoSet ELISA, and Mouse IL-12 p70 Duoset ELISA (R&D Systems, Minneapolis, MN, USA). The sensitivity was 2 pg/mL for IFN-γ and 30 pg/mL for IL-10 and IL-12. The concentrations of cytokines were calculated from the respective calibration curves obtained using the standards from these systems.

For quantification of IL-6 and IL-10, the Mouse IL-6 DuoSet ELISA and Mouse IL-10 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) were used. Cytokine quantification was performed by following the manufacturer’s instructions.