Mouse il 10 elisa set

CD4⁻ MutuDC2s and MutuDC1s were seeded at a density of 2.5 × 10⁵ cells/cm² in 96-well or 48-well plates and incubated for 24 h with 450 μL/cm² of the following TLR agonists diluted in complete medium: Pam3CSK4 (150 ng/mL, tlrl-pms, InvivoGen), poly(I:C) (8.5 μg/mL, tlrl-pic, InvivoGen), LPS from E. coli (100 ng/mL, tlrl-peklps, InvivoGen), ultrapure flagellin from B. subtilis (100 ng/mL, tlrl-pbsfla, InvivoGen), FSL-1 (100 ng/mL, tlrl-fsl, InvivoGen), Gardiquimod™ (1 μg/mL, tlrl-gdqs, InvivoGen), CpG ODN 1826 (1 μM, TriLink BIOTECHNOLOGIES). In all the experiments each condition was plated in technical triplicate. The supernatants were analyzed by ELISA for the presence of IL-6, IL-10, IL-12/IL-23 p40, IL-12p70, and MCP-1(CCL2) using the following kits according to manufacturer's instructions: Mouse IL-6 ELISA Set (555240, BD Biosciences) or Mouse IL-6 ELISA Ready-SET-Go! (88-7064, eBioscience), Mouse IL-10 ELISA Set (555252, BD Biosciences) or Mouse IL-10 (Interleukin-10) ELISA Ready-SET-Go! (88-7104, eBioscience), Mouse IL-12 (p40) ELISA Set (555165, BD Biosciences), Mouse IL-12 (p70) ELISA Set (555256, BD Biosciences), Mouse CCL2 (MCP-1) ELISA Ready-SET-Go! (88-7391, eBioscience).

The levels of IL-4, IL-5, IL-6, IL-10 and IL-13 in BALF were measured using ELISA as described previously^{38 (link)}. Briefly, antibodies 11B11, TRFK5, and eBio13A were used to capture IL-4, IL-5, and IL-13, respectively, and biotinylated antibodies BVD6-24G2, TRFK4, and eBio1316H were used for detection. The antibodies were purchased from BD Biosciences (San Diego, CA), except for eBio13A and eBio1316H, which were purchased from eBioscience. The level of IL-10 was measured using Mouse IL-10 ELISA Set purchased from BD Biosciences. The level of eotaxin 3 was measured by using a BMS5008ELISA kit (eBioscience) according to the manufacturer’s instructions. Levels of total IgE in BALF were measured using an ELISA kit 439807 (BioLegend, San Diego, CA) according to the manufacturer’s instructions.

Levels of TNF and IL-10 in the murine or human cell supernatants were determined by ELISA using the Mouse TNF ELISA Set II, the Mouse IL-10 ELISA set (BD Biosciences) and the human TNF ELISA set (Thermo Fisher Scientific), following the manufacturer’s instructions.
ROS production by memory mBMMs was assessed using MitoSOX Red (Life Technologies). Briefly, after stimulation experiments, cells were washed and stained with MitoSOX Red for 30 min at 37°C. After extensive washing with warm PBS, cells were trypsinized and fluorescence signal was acquired by flow cytometry in a BD FACS Canto II cytometer (BD Biosciences, Madrid, Spain). Phorbol-12-myristate-13-acetate (PMA) was used at 0.8 μM to increase ROS in memory macrophages 30 min prior to the second incubation with L. plantarum.

Protein concentrations of IgA, tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 in colonic tissue were measured using the Bethyl Laboratories IgA enzyme‐linked immunosorbent assay (ELISA) Kit (Bethyl Laboratories), Mouse TNF ELISA Set II (BD Biosciences; Cat.558534) and Mouse IL‐10 ELISA Set (BD Biosciences; Cat.555252) respectively, according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay (Mouse IL-10 ELISA Set; BD OptEIA, San Diego, California, USA, catalog number: 555282), 2-CAT (N-D) Research ELISA (ImmuSmol, Talence, France, catalog number: BA-E-5500) and Adrenaline/Epinephrine ELISA (LSBio, Seattle, Washington, USA, catalog number: LS-F5372) were conducted according to manufacturer’s instructions.

TNF, IL-6, and IL-10 levels were determined in cell supernatants using mouse and human TNF ELISA set II (BD OptEIA), mouse IL-10 ELISA set (BD OptEIA) and human IL-6 ELISA set (BD OptEIA), according to the manufacturer’s recommendations.

For cytokine analysis, sketched in Figure 3, the same experimental design as in vascular permeability assay was used. After dosing profiles and evaluation times were completed, the brains were dissected and individually homogenized at a 5:1 weight:volume ratio with a PBS solution at pH 7.4 with 1% phenyl methyl sulfonyl fluoride (PMSF), using an ULTRA-TURRAX T25 basic polytron at 6500 rpm. After homogenization, the samples were centrifuged at 14,000 rpm for 7 min, the supernatant was recovered for cytokine quantification by the ELISA method using cytokine kits (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions for IL-10 and TNF-α: Mouse IL-10 ELISA Set (Cat. No. 555252) and Mouse TNF Mono/Mono ELISA Set (Cat. No. 555268), respectively.