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Cytokines (IL-17A/F, IL-21, IL-22, IL-23A, IL-6, IL-1β, IFN-γ and TGF-β) were measured in duplicate by ELISA in culture supernatants from unstimulated and antigen stimulated PBMCs of leprosy patients (Ready Set Go, e-Bioscience, San Diego, CA, USA) as per manufacturer’s instructions. In brief, 96-well plates (Nunc, Rochester, NY, USA) were coated overnight at 4°C with biotin conjugated anti human antibodies for each of the cytokines, Plates were washed 5 times, blotted and wells blocked with assay diluents for 1 h at room temperature. 100ul/well of culture supernatant was added and plates incubated overnight at 4°C. After washing with buffer, avidin-horseradish peroxidase-conjugated anti-mouse antibody was added and the plates incubated at room temperature for 30 min. Subsequent to washing wells as before, color development was undertaken using TMB (Tetramethylbanzedine) substrate and the reaction stopped by 1 N H₂SO₄. The optical density (OD) of each well was read at 450 nm.

Caco2 cells were plated at 900,000 per well in 24-well plates. Upon attachment to the plate, cells were treated as described in the text and supernatant was harvested after 24 h. Experiments with cells were performed four times and experiments with organoids were performed nine times. Cytokine levels in supernatants from intestinal cells and organoids were determined by ELISA (Ready-SET-Go!^® eBioscience, San Diego, CA, USA) as per manufacturer’s instructions. All samples were tested in duplicate in the ELISAs.

Nitric oxide metabolites released in the cell culture supernatant as nitrate or nitrite was detected by Total Nitric Oxide detection kit (Thermo-Scientific) according to the manufacturer’s protocol. Photometric measurement of the absorbance due to this azo chromophore determined the NO₂^- (nitrite) concentration at 540 nm wavelength using an ELISA plate reader (BioTek Instruments).
Cytokine ELISA of TNFα, IL-1, IL-6, IL-10, IL-12, and TGFβ was done using specific antibodies (Ready-SET-Go!, eBioscience, Thermo-Scientific). Optical densities were measured using an ELISA plate reader at 450 nm wavelength (BioTek Instruments, Inc).

Peritoneal macrophages were plated in a 96-well plate at the concentration of 1 × 105 cells/well and kept for 24 h in incubator at 37 °C with 5% CO2, for cell adhesion. The next day, the culture medium was exchanged, and the cells were stimulated with the peptide (6.25 μM and 25 μM) and/or LPS (500 ng/mL) with or without previous stimulation After the whole period of interaction, the supernatant was recovered and stored at -20 °C for cytokine dosage. In all assays of cytokine dosage, the samples were stored at − 20 °C. Cytokine levels were measured by ELISA (Enzyme-Linked Immunosorbent Assay) using Ready-SET-Go! ®(eBioscience™, Inc., Affymetrix, USA), following the manufacturer’s protocols.

Naive CD4⁺ T cells were obtained by incubating single-cell suspensions of spleen and lymph nodes with biotinylated antibodies to CD8, CD16, CD19, F4/80, Gr-1, MHC class II (I-Ab), CD11b, CD11c, and DX5 followed by incubation with Streptavidin Microbeads (MACS, Miltenyi Biotec). CD4⁺ T cells were isolated by negative selection in an auto-MACSTM Pro Separator (Miltenyi Biotec). Next, cells were activated with plate-bound anti-CD3 (5 µg/ml) and anti-CD28 (2 µg/ml) in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS, 2 × 10⁻³ M l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol, and the corresponding cytokine cocktail: for Th0, anti-IFNγ (4 µg/ml), anti-IL-4 (4 µg/ml), and IL-2 (10 ng/ml); for Th1 anti-IL-4 (4 µg/ml), IL-12 (10 ng/ml), and IL-2 (10 ng/ml); for Th17 anti-IFNγ (4 µg/ml), anti-IL-4 (4 µg/ml), IL-6 (20 ng/ml), IL-23 (10 ng/ml), and TGF-β1 (5 ng/ml); and for Treg anti-IFNγ (4 µg/ml), anti-IL-4 (4 µg/ml), and TGF-β1 (10 ng/ml). After 72 h of culture, IFNγ, IL-17, or IL-10 in the supernatant were measured by ELISA (Ready-SET-Go, eBiosciences). For FACS analysis, intracellular cytokine staining was preceded by restimulation for 4 h with 50 ng/ml phorbol dibutyrate (PMA) and 500 ng/ml ionomycin in the presence of brefeldin A (1 µg/ml) (BD Biosciences).