Cytometric bead array flex set kit

Plasma cytokines and chemokines were quantified with Becton-Dickinson Cytometric-Bead-Array (CBA) FlexSet kits, according to the manufacturer’s protocol. The standard sensitivity array (picograms/mL) included interleukin-4 (IL-4), chemokine CxCL10, interleukin-5 (IL-5), and transforming growth factor-β (TGF-β). The enhanced sensitivity array (femtograms/mL) included interferon-ɣ (IFN-ɣ), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12p70 (IL-12p70), interleukin-17A (IL-17A), interleukin-10 (IL-10), and tumor necrosis factor- α (TNF-α). Raw data were collected on an Accuri C6 Plus cytometer (BD-Biosciences, San Jose, CA, USA) and analyzed with FlowJo version-10 (TreeStar, Ashland, OR, USA).

Whole blood samples were obtained from all children. All samples were obtained between 10:00am and 4:00pm. After collection, blood was allowed to clot by leaving it undisturbed at room temperature and then serum extracted after blood had been processed at 1,000–2,000 x g for 10 minutes in a refrigerated centrifuge. Serum was kept at −80°C until further analyzed. As the samples were labeled with numbers, without any group identification, the investigators were blinded for all procedures.
BDNF serum levels were measured with sandwich-ELISA, using a commercial kit according to the manufacturer's instructions (Milipore, USA). For assessment of oxidative stress, serum levels of malondialdehyde (MDA), a product of lipid peroxidation, were measured by the TBARS (thiobarbituric acid reactive substances) method [51 (link)]. Serum IL6 levels were measured by flow cytometry using the Cytometric Bead Array (CBA) Flex Set Kit (BD Biosciences, San Jose, CA) (Cat. #558276). Acquisition was performed with a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The instrument has been checked for sensitivity and overall performance with Cytometer Setup and Tracking beads (BD Biosciences) prior to data acquisition. Quantitative results were generated using FCAP Array v1.0.1 software (Soft Flow Inc., Pecs, Hungary).

The excised spleens were crushed through a 70 μm cell strainer (BD labware, New Jersey, USA) to obtain single cell suspensions as described previously [38 (link)]. Cell numbers were determined with a Cell Coulter Z1 (Beckman Coulter Inc., Florida, USA). Cells were cultured in medium (RPMI 1640 with L-glutamine and 10 % foetal bovine serum and 1 % streptomycin/penicillin) in 96 well plates at a concentration of 2.7 × 10⁶ cells/ml per well at 37 °C in a humidified atmosphere with 5 % CO₂ for three days with concanavalin A (ConA) (5 μg/ml), and for five days with or without Lupex or trypCry1Ab (17.2 μg/mL). The total volume in each well was 200 μl. The amount of cytokine IL-2, IL-5, IL-13, IL-10, and interferon gamma (IFNγ) released into spleen cell supernates were determined by Cytometric Bead Array (CBA) flex set kit from BD Biosciences (San Diego, California, USA).

SI‐LP, LI‐LP and MLN cell suspensions were re‐stimulated in vitro with PMA (50 ng/mL, Sigma‐Aldrich) and Ionomycin (500 ng/mL, Sigma‐Aldrich) for 4 hours. At 1 hour of re‐stimulation, cells were treated with Brefeldin A (3 μg/mL, BioLegend). For cDC cultures, FACS‐sorted cDC (2 × 10⁴ cells/well) were incubated in R10 medium, in the presence or absence of LPS (1 μg/mL, Sigma‐Aldrich) or FliC (100 ng/mL;) for 22 hours at 37°C and 5% CO₂. Cells were analysed by flow cytometry, and levels of IL‐6 in cell supernatants assessed using the cytometric bead array (CBA) flex set kit (BD Bioscience) according to manufacturer's instructions.

CCL18 was quantified using a DuoSet ELISA Development System (R&D Systems Europe, UK). IL-6 concentrations were measured by flow cytometry using the corresponding Cytometric Bead Array Flex Set kit (BD Biosciences).