Fcap array

The cytokines, IL-2, IFN-γ, IL-6, TNF-α, IL-4, IL-10, and IL-17, present in culture supernatants were simultaneously quantified by the CBA technique (BD ™ CBA Human Th1/Th2/Th17 Cytokine Kit, San Jose, CA, USA) according to the manufacturer’s protocol. Then, the beads bound with the cytokines were resuspended in 200 µL of the wash buffer and transferred to cytometry tubes for acquisition that was made on the same day on the FACSCalibur BD flow cytometer (BD Biosciences, USA). The acquisition analysis was performed using the FCAP Array^TM version 2.0 program (BD Biosciences, USA), and the concentration of cytokines was estimated by linear regression analysis with the fluorescence obtained on the standard curve of each cytokine and expressed in pg/mL.
For this analysis, 10 individuals from the CG, 10 patients from the SD group, and 9 from the IB group were selected, and the levels of cytokines of the three stimulus conditions, namely αCD3αCD28, STAg, and absence of stimulus, were measured.

Key human inflammatory cytokines were assessed within mucosal biopsies via Cytometric Bead Array as previously described (17 (link)). IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ were assessed using a Human T_h1/T_h2/T_h17 Cytokine Kit (BD Biosciences). Flex set kits were also selected for an additional three cytokines of interest (IL-5, IL-8, and IL-13). In brief, tissue biopsies were first weighed and then added to lysis tubes with 2 mm ceramic beads and 200 μL PBS, and ruptured in a bead ruptor 24 (Omni International) at 3.55 m/s for two cycles of 10 s. The homogenate was centrifuged at 1,500 × g for 5 min, and then 25 μL of the supernatant was transferred to assay tubes. Cytokine analysis on the supernatant was conducted as per the manufacturer's instructions. Data were acquired on an LSR II with BD FACSDiva Software (version 6.1.3) and analyzed using FCAP Array^TM (version 1.0.1) (BD Biosciences). A 5-parameter logistic model was used to produce standard curves with a fitting accuracy of >99.9% for all cytokines. Values below the threshold for the limit of detection for each cytokine were recorded as 0. IL-13 was below the limit of detection for all samples and was excluded from subsequent analyses. Final concentrations were normalized to concentration per gram of tissue based on the initial tissue weight.