Human or murine blood monocytes were collected and purified using gradient Ficoll-Hypaque (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and the Negative Selection Monocyte Isolation kit (STEMCELL Technologies, Vancouver, Canada). The fresh isolated monocytes were cultured in RPMI1640 medium (HyClone, Logan, UT, USA) supplemented with 10% FBS (PAN Biotech, Aidenbach germany), 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA) at 37°C in a 5% CO2 incubator. To further test the effect of TNF-α and IL-6 on PPARa expression, monocytes were stimulated with 50ng/ml GM-CSF (BioLegend, San Diego, CA, USA), and different dose of TNF-α and IL-6 (Biolegend, San Diego, CA, USA) for 7 days. The macrophages cell lines RAW264.7 and MSC2 were also cultured in RPMI1640 complete medium.
Tnf α and il 6
Manufactured by BioLegend
Sourced in United States
TNF-α and IL-6 are cytokines that play important roles in immune system regulation and inflammatory processes. TNF-α is a pro-inflammatory cytokine, while IL-6 has both pro-inflammatory and anti-inflammatory functions. These cytokines are commonly used in research applications to study immune and inflammatory responses.
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4 protocols using tnf α and il 6
1
Monocyte Isolation and Functional Assay
2
Dectin Receptor Modulation of pDC Responses
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The pDCs were cultured in 96-well plates in pDC media. The cells were left untreated or incubated with anti-dectin-1, dectin-2, dectin-3, or antimincle antibodies (100 µg/mL) for 30 min at 37°C. The pDCs were then challenged with P. brasiliensis yeast cells at a final volume of 200 µL in pDC media at the ratios indicated in the figure legends. Control wells contained pDCs only, pDCs and antibodies, pDCs and CpG, or pDCs and LPS. For the inhibition of TLR-9, pDCs were left untreated or incubated for 30 min with either 10 µg/mL of bafilomycin A1 or 10 µg/mL of the TLR-9 antagonist A151. After 18 h of incubation at 37°C, the supernatants were removed and the levels of TNF-α, IL-6, IL-1β, IFN-α, and IFN-β were measured by ELISA according to the manufacturers’ protocols (eBioscience for TNF-α and IL-6; Biolegend for IL-1β; PBL Assay Science for IFN-α and IFN-β).
3
Serum Cytokine Profiling in Mice
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Mice were cheek-bled prior to sacrifice, and serum was isolated. TNFα and IL-6 (Biolegend, San Diego, CA, USA) and IL-1β (R&D Systems, Minneapolis, MN, USA) were measured by ELISA.
4
Cytokine Quantification by ELISA
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TNFα, IL-1β and IL-6 were measured using ELISA kit according to the manufacturer protocol (TNFα and IL-6: BioLegend; IL-1β: R&D). Cells were lysed with ddH2O. Supernatants and lysates were collected and stored at -80 °C until assayed.
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