A. fumigatus conidia (5 × 104) were plated in 96-well plates and grown in pDC media to hyphae of 10–20 μm average length. pDCs (5 × 104) were left untreated or incubated with anti-Dectin-2 or anti-Dectin-1 antibodies for 30 minutes at 4°C. pDCs were then added to the hyphae in a final volume of 200 μl pDC media containing voriconazole (0.5 μg/mL) to inhibit fungal overgrowth. Control wells contained pDCs only, pDCs and antibodies, or pDC and CpG. After 6 hr of incubation at 37°C, the supernatants were removed and TNF-α and IFN-α levels were measured by ELISA according to the manufacturers’ protocols (eBioscience for TNF-α; PBL Assay Science for IFN-α).
Tnf α
Manufactured by PBL Assay Science
The TNF-α assay is a laboratory tool used to measure the concentration of the cytokine tumor necrosis factor alpha (TNF-α) in various biological samples. TNF-α is a signaling protein involved in inflammatory processes and immune function. This assay provides a quantitative analysis of TNF-α levels, which can be useful for research applications.
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Lab products found in correlation
Ifn γ, by PBL Assay Science (2 mentions)
Ifn β, by PBL Assay Science (1 mentions)
Puromycin, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Nf κb p65 sc 372, by Santa Cruz Biotechnology (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by GE Healthcare (1 mentions)
Human recombinant ifnβ, by PBL Assay Science (1 mentions)
Dmxaa, by Apexbio (1 mentions)
Human s386 phospho irf3, by Abcam (1 mentions)
Ifit1 isg56, by Thermo Fisher Scientific (1 mentions)
Sting, by Cell Signaling Technology (1 mentions)
Polybrene, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
3 protocols using tnf α
1
Dectin-2 Modulates Antifungal Immunity
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A. fumigatus conidia (5 × 104) were plated in 96-well plates and grown in pDC media to hyphae of 10–20 μm average length. pDCs (5 × 104) were left untreated or incubated with anti-Dectin-2 or anti-Dectin-1 antibodies for 30 minutes at 4°C. pDCs were then added to the hyphae in a final volume of 200 μl pDC media containing voriconazole (0.5 μg/mL) to inhibit fungal overgrowth. Control wells contained pDCs only, pDCs and antibodies, or pDC and CpG. After 6 hr of incubation at 37°C, the supernatants were removed and TNF-α and IFN-α levels were measured by ELISA according to the manufacturers’ protocols (eBioscience for TNF-α; PBL Assay Science for IFN-α).
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A. fumigatus conidia (5 × 104) were plated in 96-well plates and grown in pDC media to hyphae of 10–20 μm average length. pDCs (5 × 104) were left untreated or incubated with anti-Dectin-2 or anti-Dectin-1 antibodies for 30 minutes at 4°C. pDCs were then added to the hyphae in a final volume of 200 μl pDC media containing voriconazole (0.5 μg/mL) to inhibit fungal overgrowth. Control wells contained pDCs only, pDCs and antibodies, or pDC and CpG. After 6 hr of incubation at 37°C, the supernatants were removed and TNF-α and IFN-α levels were measured by ELISA according to the manufacturers’ protocols (eBioscience for TNF-α; PBL Assay Science for IFN-α).
2
Multiplex Cytokine Quantification and CyTOF Analysis
3
Immune Signaling Pathway Activation Assay
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Dimethyl sulfoxide was obtained from Thermo Fisher. Puromycin was obtained from Clontech and used at 3 μg/ml in cell culture medium. LPS and Polybrene were obtained from Sigma-Aldrich. Human recombinant IFN-β, IFN-γ, universal mammalian IFN, and TNF-α were obtained from PBL. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were obtained from Gemini Bio-Products. Steady-Glo cell lysis/luciferin reagent was obtained from Promega. Poly(I-C) was obtained from GE Healthcare. Stocks of G10 were synthesized by the OHSU Medicinal Chemistry Core Facility. DMXAA was purchased from ApexBio. Antibodies against the following antigens were used (with the source indicated in parentheses): glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-51906; Santa Cruz Biotechnology); IRF3 (sc-9082; Santa Cruz Biotechnology); human S386 phospho-IRF3 (catalog number 2562-1; Epitomics); STING (catalog number 3337; Cell Signaling); IPS-1 (A300-782A; Bethyl); IFIT1/ISG56 (PA3 848; Thermo Fisher); TRIF (4596; Cell Signaling); Mx1 (GTX111153; GeneTex); β-actin (MAB0501R; Thermo Fisher); IκBα (Santa Cruz sc-371; Santa Cruz Biotechnology); NF-κB P65 (sc-372; Santa Cruz Biotechnology).
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