Quantitative elisa

The serum and fecal concentrations of the biomarkers that were initially established to be important in the pathogenesis of IBS (MCP-1, MIP-1β, TNF-α, IFN-γ, IL-1β, IL-10, IL-4, IL-13, and CXCL16), based on the initial data from the Proteome Profiler Human Cytokine Array assay, were determined by quantitative ELISA (R&D Systems, Minneapolis, MN). The instructions provided by the manufacturer were followed in determining the serum concentrations of each of these biomarkers. The sensitivity and the assay range, respectively, of the ELISA test for each of the biomarkers tested are as follows: MCP-1 (10 pg/mL, 31.2–2,000 pg/mL); MIP1-β (11 pg/mL, 15.6–1,000 pg/mL); IFN-γ (8 pg/mL, 15.6–1,000 pg/mL); TNF-α (5.5 pg/mL, 15.6–1,000 pg/mL); IL-1β (1 pg/mL, 3.9–250 pg/mL); IL-10 (3.9 pg/mL, 7.8–500 pg/mL); CXCL16 (0.017 ng/mL, 0.156–10 ng/mL).
For the fecal concentrations, stool samples (200 mg) from 20 healthy volunteers, 20 ID-IBS, and 10 PI-IBS were suspended in 500 μL of PBS and centrifuged at 10,000×g for 10 min to remove the debris and solid materials in present. The supernatant fluids were tested for the biomarkers listed above according to the manufacturer's instructions.

To measure the concentrations of MMP2 and MMP9 secreted from the cultured tumor cell lines, the supernatants were assessed using ELISA. Cells (5×10⁴/well) in 24-well plates were incubated at 37°C in a 5% CO₂ atmosphere in DMEM containing 10% FBS. After 24 h, the cells were washed and incubated for 24 h in serum-free medium without/with Salin (5–20 μmol/l). The culture-conditioned medium was collected, centrifuged, and the concentrations of MMP2 and MMP9 were determined by quantitative ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Tumor cells were pre-treated with 0.1 μM I3A or DMSO control for 48 h, then washed and plated in a 24 well plate for 4 h to adhere, followed by addition of T cells. 2.5 × 10⁵ T cells were added to 5 × 10⁵ tumor cells treated with I3A or DMSO. Approximately 24 h post-coculture, supernatant was collected and frozen for subsequent analysis. IFNγ and IL-2 production were measured using a quantitative ELISA per the manufacturer's instructions (R&D Systems, Minneapolis, MN).

T cells were cultured alone or with LM7 tumor cells at a 1:1 effector to target ratio without the provision of exogenous cytokines. Approximately 24 hours after coculture initiation, supernatant was collected and frozen for later analysis. Production of IFNγ and IL2 was measured using a quantitative ELISA per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

The secretome of encapsulated ASCs after 5 days of culture was compared to the secretome of ASCs grown as a confluent monolayer. Encapsulated and monolayer ASCs were incubated in serum-free-medium, supernatant was sampled at 48 h, and frozen at −80 °C. IL-6, IL-8, and VEGF were assayed by quantitative ELISA (R&D system, Minneapolis, MN, USA) in supernatants from cell cultures for seven donors at P3 with one donor at P3 and P4. A qualitative ELISA (Multi-Analyte ELISArray Kits, QIAGEN, Hilden, Germany) detecting 12 cytokines: IL-1β, IL-4, IL-6, IL-10, IL-12, IL-17A, IFNγ, TNFα, TGF-β1, MCP1, MIP 1α, and MIP 1β, was also performed on supernatants from four donors at P3. Experiments were performed on three samples from each donor.