Mouse th1 th2 th17 cytometric bead array kit

For this study, two blood samples were collected: the first from the retro-orbital region after 15 days of treatment and the second after the second treatment, coinciding with the euthanasia of the animals. Cytokine levels were determined by flow cytometry using the Mouse Th1/Th2/Th17 Cytometric Bead Array kit (BD Biosciences, San Jose, CA, USA), following the manufacturer’s instructions. The quantification of cytokines included interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFN-γ), tumour necrosis factor (TNF), interleukin-17A (IL-17A), and interleukin-10 (IL-10). Samples were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, USA) and analyzed with FCAP Array v3.0 Software (BD Biosciences, San Jose, CA, USA).

Single cell suspensions of spleen cells were seeded into anti-CD3 antibody-coated plates. After 3 days, culture supernatants were collected and their cytokine content was measured using the mouse Th1/Th2/Th17 cytometric bead array kit as instructed by the manufacturer (BD Biosciences; Cat. No. 560485). Cytokines that were measured included IL-2, IFN-γ, IL-6, IL-17A, TNF-α, IL-4, and IL-10. The levels of each cytokine were measured by flow cytometry and analyzed using FCAP Array software (Soft Flow, Inc., St. Louis Park, MN, USA).

The levels of TNF-α, IL-6, IFN-γ, IL-2, IL-4, IL-10, and IL-17α were determined in plasma samples through the Mouse Th1/Th2/Th17 Cytometric Bead Array kit (560485, BD Biosciences, San Jose, CA). Bead fluorescence was measured using the BD Biosciences LSRII flow cytometer and analyzed with the accompanying software. Protein levels of CCL5, CCL11, CCL17, CXCL1, CXCL9, CXCL10, CXCL13, CXCL5, CCL22, CCL2, CCL3, and CCL4 in tissue and mast cell supernatants were analyzed with the LEGENDplex Mouse Proinflammatory Chemokine Panel (740007, Biolegend) per the manufacturer's instructions. Supernatants were loaded without dilution. Hundred micrograms of tissue lysate were loaded for analysis of chemokine levels in the skin, although final values displayed in the paper were normalized to nalyseds per milliliter per microgram of protein loaded. Bead fluorescence was measured using the BD Biosciences LSRII flow cytometer and analyzed in the Biolegend LEGENDplex data analysis software.

Cytokine levels were quantified (mouse Th1/Th2/Th17 cytometric bead array kit, BD Biosciences) and analyzed (FACS Canto, BD Biosciences)/FCAP Array Software (Soft Flow Hungary Ltd.).

To assess the impact of modulating IL-35 on the cytokine environment during P. berghei infection, cytometric bead array procedure for the simultaneous detection of IL-10, IL-17A, TNF, IFN-γ, IL-6, IL-4 and IL-2 was performed on serum samples collected from uninfected control mice and P. berghei infected mice on the 5th day following P. berghei infection after IL-35 modulation treatment. The assay was performed using a commercially procured mouse Th1/Th2/Th17 cytometric bead array kit (BD biosciences, San Jose, CA, USA). Staining procedure(s) were conducted as described (Instruction manual BD CBA Mouse Th1/Th2/Th17 cytokine kit, catalogue no. 560485). Sample acquisition was achieved using a BD LSR Fortessa fluorescence activated cell sorter via phycoerythrin (PE) and allophycocyanin (APC) channels. Prior to sample acquisition, gating layouts were generated for 2400 events from unstained control tubes and data acquired was analysed with FCAP Array software version 3.0 (BD Bioscience, San Jose, CA, USA).

The levels of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 were determined using a mouse Th1/Th2/Th17 cytometric bead array kit (BD Biosciences). Levels of MCP-1, GM-CSF, G-CSF, MIP-1α, MIP-1β, MIG, and RANTES were determined using cytometric bead array flex sets. Relative amounts of each cytokine/chemokine were analyzed using a FACSCanto (BD Biosciences) flow cytometer and FCAP Array Software (Soft Flow Hungary Ltd. for BD Biosciences, St. Louis Park, MN, USA).

Splenocytes from immunized mice were cultured (1 × 10⁶ cells/well) with 5 µM of pooled HIV-1 peptides for five days under an atmosphere of 5% CO₂ at 37°C. Supernatants were harvested, and cytokines were detected using a Mouse Th1/Th2/Th17 Cytometric Bead Array Kit (BD) according to the manufacturer’s instruction.