Sandwich elisa kit

BEAS-2B cells (2 × 10⁵ cells/mL) were treated with 0–30 μM fisetin at 37 °C for 1 h and then incubated with 10 ng/mL TNF-α or 10 ng/mL TNF-α/IL-4 at 37 °C for 24 h. The supernatants were collected to detect the levels of intercellular adhesion molecule-1 (ICAM-1), IL-8, IL-6, MCP-1, and c-c motif chemokine ligand CCL5, CCL11, and CCL24 using sandwich ELISA kits (R&D, Minneapolis, MN, USA). Specific protein levels were detected using a microplate reader (Thermo) at an OD of 450 nm as described previously [25 (link)]. Fisetin (10 μM) was combined with MAPK specific inhibitors, including 10 μM SP600125 (JNK inhibitor), 10 μM SB203580 (p38 inhibitor), and 10 μM PD98059 (ERK inhibitor) (Enzo Life Sciences, Inc., Farmingdale, NY, USA), to detect the ICAM-1 protein level by ELISA.

The capacity of ES L1-stimulated DCs to instruct the polarization of T cells was analyzed in allogenic stimulation assay. DCs (0.5 × 10⁴/well, 96-well round-bottom plate) were cultivated with naive allogenic T lymphocytes in 1:20 ratio, for 6 days. To detect cytokines in the supernatants of allogenic stimulation assay, DC/T cell cocultures were treated with PMA (20 ng/ml) and Ionomicin (500 ng/ml) for the last 4 h of coculture, followed by the harvesting of cell-free supernatants. The levels of cytokines produced in DCs cultures (IL-10, IL-12p70, TGF-β), as well as in DC/T cell cocultures (IL-4, IL-10, IFN-γ, IL-17, TGF-β), were measured in cell-free supernatants by sandwich ELISA Kits (R&D). Additionally, T cells were primed with DCs (2 × 10³/well of 96-well round-bottom plate) at a 1:50 DC/T cell ratio for 3 days and then expanded with IL-2 (2 ng/ml, R&D) for two more days (T primed cells, Tpr). In some experiments 1-methylthryptophane (1-MT, 0.5 mM, Sigma), as an IDO-1 inhibitor (48 (link)), was added in the priming cocultures to assess the role of IDO-1 in the induction of Tregs. The Tregs were identified by flow cytometry based on their expression of CD4, CD25, FoxP3, IL-10, and TGF-β by flow cytometry.

HDFs were plated in 48-well plates (2 × 10⁴ cells/well) and incubated for 24 h. Next, the medium was discarded, replaced by serum-free DMEM, and the plates were incubated overnight. The cells were then treated with the indicated concentrations of the samples for 1 h and subsequently with 20 ng/mL TNF-α for 12 h (to test the secretion of IL-1β and IL-6) and 24 h (to investigate MMP-1 and COLIA1 secretion). The concentration of IL-1β, IL-6, MMP-1, and COLIA1 in the supernatant were determined using sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA).

Fasting blood sugar (FBS) and hemogloblin A1c (HbA1c) assays were performed on participants. SPINREACT (Girona, Spain) and MyBiosource (California, USA) commercial kits were used. The C-peptide ELISA kit was provided by DRG Diagnostics (Marburg, Germany). Total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein-cholesterol (HDL-C) levels were measured using a colorimetric enzymatic method (Spain). The Friendewald et al. [20 (link)] formula was used to estimate the low-density lipoprotein-cholesterol (LDL-C) levels. Sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to detect IFN-γ and IL-15 levels in the blood. With each kit, all steps were completed according to the manufacturer’s recommendations.

Plasma levels of pro-inflammatory cytokines TNFα, IL-1β, and IL-6 were analyzed with commercially available sandwich ELISA kits (R&D Systems, Minneapolis, MN). High-sensitivity C-reactive protein and insulin were measured using chemiluminescence-based Immulite immunoassay systems (Diagnostic Products Corporation, Los Angeles, CA). Plasma glucose concentrations were measured by calorimetric method in the CTRC Core Laboratory and whole blood hemoglobin A1c was measured by the OHSU Hospital Clinical Laboratory. The OHSU Lipid Laboratory analyzed lipoproteins using beta quantification enzymatic methods that determine total cholesterol, triglyceride levels, HDL cholesterol, and calculated VLDL cholesterol, LDL cholesterol, and non-HDL cholesterol. In samples where plasma triglyceride levels exceeded 300 mg/dL, lipoproteins were separated by preparative ultracentrifugation before analysis.

Confluent layers (10⁵ cell/well) of HUVECs were cultured in 96-well plates for 24 hours in 100 µl Comp-AIM-V medium in the presence of rMASP-1 or other endothelial cell activating factors. IL-1alpha, IL-1ra, IL-6, IL-8, MCP-1, and TNFalpha were measured by sandwich ELISA kits according to the manufacturer's protocol (R&D Systems). We also analyzed the production of IL-6 and IL-8 at 1, 3, 6, 10, and 24 hours.