Cytokine and chemokine protein expression were analyzed by multiplex assay using the Human Cytokine/Chemokine Magnetic Bead Panel (Luminex, #HCYTMAG-60K-PX30) on a MAGPIX system (Merck). Fold changes relative to the corresponding control were calculated and plotted as log2FC. Lower and upper limits of quantitation (LLOQ/ULOQ) were inputted from standard curves for cytokines above or below detection. The media from control cells and those treated with ADU-S100 (Chemietek) were subjected to CXCL10 ELISA assay (R&D systems, #DIP100) at 24 h, whereas in TBK1/IKKε studies supernatants were analyzed at 48 h. Additionally, supernatants from untreated and ADU-S100 treated samples were subjected to CCL5 and IFNβ ELISA assays (R&D systems, #DRN00B, #DIFNB0) at 72 h. Assays are representative of 3 experiments in duplicate.
Drn00b
Manufactured by R&D Systems
Sourced in United States
The DRN00B is a laboratory equipment product designed for research and development applications. It serves as a core function for various experimental processes. The detailed specifications and intended uses of this product are not available at this time.
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6 protocols using drn00b
1
Multiplex Cytokine and Chemokine Analysis
2
Quantifying Serum CCL5 Levels
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The concentrations of CCL5 found in blood serum were determined using a commercially available ELISA kit (Cat. n#DRN00B, R&D systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Samples were initially diluted 1/100, and CCL5 concentration was calculated by interpolation into a standard curve. CCL5 values are expressed as ng/mL. Intra-assay and inter-assay precision are coefficient of variation (CV) < 2.4% and CV < 6.5%, respectively.
3
Quantification of Immune Mediators
4
Analysis of Inflammatory Cytokines
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Conditioned medium collected from 3D models in the absence or presence of anakinra was subjected to ELISA for CXCL8, CCL2 (BD Biosciences 555244 and 559017, respectively) and CCL5 (R&D systems, DRN00B) according to the manufacturer's instructions.
5
Evaluating Anti-Inflammatory Cytokine Inhibition
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To evaluate the efficacy of inhibiting the secretion of inflammatory cytokines and chemokines, A549 cells were seeded in 6-well plates at a density of 5 × 105 cells/well. After replacing the medium with serum-free media (SFM), the cells were stimulated with 0.5 ng/mL IL-1β. The experimental group was treated with uRO-w (12.5, 25, 50, 100, 200 μg/mL) or EA (1.25, 2.5, 5 μg/mL) or UA (1.25, 2.5, 5 μg/mL), and the positive control group was treated with 2 μM dexamethasone (DEX; Sigma-Aldrich, St. Louis, MO), and the culture was maintained for 24 h. The cell culture supernatants were collected and analyzed for IL-8, MCP-1, RANTES, and IL-6 levels using sandwich ELISA kits (D6050, D8000C, DCP00, and DRN00B; R&D Systems).
6
Measuring Cytokine Secretion in OSA Cells
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Supernatant from OSA cells incubated with 10 μg/ml 2’3’-cGAMP or control media for 24 hours was collected and centrifuged at 2000 g for 10 minutes at 4°C, aliquoted, and frozen at -80°C. The concentrations of CXCL10 and CCL5, were measured using human CXCL10/IP-10 ELISA kit (Quantikine, DIP100, R&D Systems, Minneapolis, MN, USA) and human CCL5/RANTES ELISA kits (Quantikine, DRN00B, R&D Systems, Minneapolis, MN, USA), respectively. Assays were performed in duplicate per manufacturer protocols, and 2–3 independent replicates were performed.
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