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45 protocols using recombinant tnf α

1

Cytokine-induced Transcriptional Profiling

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A549 cells were stimulated with recombinant TNF-α (Gibco), IFN beta (PBL Assay Science), IFNL1 (PBL Assay Science), or different combinations of these cytokines for 8 h at 100 U/ml in EMEM supplemented with 5% NaHCO3, penicillin-streptomycin (Gibco), l-glutamine (Gibco), and 1% FBS. Also, some cells were infected with PR8-NS1-GFP at an MOI of 2 as a positive control. Cells were lysed and stored at −80°C for subsequent RNA extraction and qRT-PCR analysis.
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2

In vitro model of IVH-induced inflammation

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HCPEpiCs (2 × 105/well) were then treated in BioLite 6-well plates (Thermo Scientific, Rochester, NY, USA) with metHb, ferrylHb, heme (25–50 µM), or recombinant TNF-α (100 ng/mL, Gibco, Carlsbad, CA, USA) as control for 24 h to replicate IVH-induced inflammatory cellular conditions in vitro. MetHb and ferrylHb were prepared from fresh peripheral blood samples obtained from healthy volunteers, as we have previously described [27 (link)]. After treatment, supernatants were collected and stored at −80 °C before RNA isolation and measurement of protein biomarkers. To analyze the degree of cell death caused by treatments, LDH leakage was determined in the supernatants of stimulated HCPEpiC cell cultures via photometric measurement of catalytic LDH activity on a Cobas® 6000 analyzer (Roche Diagnostics, Mannheim, Germany).
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3

Primary Mouse Hepatocyte Isolation

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Primary mouse hepatocytes were isolated from mice by perfusion with collagenase type I, as previously described18 (link). The hepatocytes were cultured overnight, followed by treatment with recombinant TNFα (GIBCO BRL, Gaithersburg, MD, USA).
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4

Evaluation of CFTR Modulators in Vitro

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CFTR correctors lumacaftor (VX-809, LUM) (S1565) and tezacaftor (VX-661, TEZ) (S7059), CFTR potentiator ivacaftor (VX-770, IVA) (S1144), voltage-independent selective CFTR inhibitor CFTRinh172 (S7139), CFTR activator Forskolin (FSK, S2449), and NF-κB pathway inhibitor BAY 11-7082 (S2913) were purchased from Selleck Chemicals (Houston, TX, United States). cAMP phosphodiesterase inhibitor IBMX (3-isobuthyl-1-methylxanthine, I5879) was ordered from Sigma-Aldrich (St. Louis, MO, United States). Except for recombinant TNF-α (Gibco, Carlsbad, CA, United States), all reagents were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich).
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5

Evaluating HCQ's Effects on RVECs

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Primary HRVECs were obtained from ScienceCell (Carlsbad, CA) and cultured with ECM medium (Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Shanghai, China) containing growth factors and 5% FBS in a humidified incubator at 37°C with 5% CO2. Cell Counting Kit-8 (CCK-8) assay kit (Bimake, Shanghai, China) was used to measure the effects of HCQ on the cell viability of RVECs: RVECs were seeded in 96-well plates with HCQ (0, 20, 40, 80 and 100 µM) for 48 h. At that time, cells were added with CCK-8 reagent diluted at a 10:1 ratio and cultured for another 2 hours before the OD value of each well was measured at 450 nm using a Synergy H1 microplate reader (BioTek, Vermont, USA). Cells were pretreated with HCQ at nontoxic concentrations for 10 h at 80%–90% confluence, and then incubated with recombinant TNF-α (10 ng/mL, Life Technologies, Carlsbad, CA) for different time according to experimental requirements.
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6

Anticoagulant Factors Evaluation

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Recombinant TNF‐α was purchased from Life Technologies. Rivaroxaban and dabigatran (Alsachim) were weighed and dissolved in DMSO; aliquots were stored at −20°C. Recombinant hirudin was dissolved in H2O and stored at −20°C (LabNed). PPACK was obtained from Calbiochem (Merck Biosciences). Plasma‐derived α‐thrombin and corn trypsin inhibitor (CTI; final concentration 70 µg/ml) were obtained from Haematologic Technologies.
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7

Metformin Modulates Inflammatory Cytokines

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After 12-hour pretreatment with 5 mM metformin, hRVECs were exposed to 2.5 ng/mL recombinant TNFα (Life technologies, Grand Island, NY) for another 12 hours. The protein concentrations of MCP-1 and IL-8 in hRVECs conditioned media were determined using ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
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8

Murine Model of Respiratory Infections

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Mice were treated i.n., i.t. or i.pls. with 20 µg LPS (Sigma Aldrich, St. Louis, MO, USA), 100 ng recombinant TNFα (Peprotech, Rocky Hill, NJ, USA), 100ng recombinant GM-CSF (Peprotech), 2.5, 5 or 10 × 106 CFU Escherichia coli (ATCC) or 7.5x106 PFU HSV-1 in a volume of 15µl saline. For talc experiments, 200 µl of a 5% Talc 0.9% NaCl solution was injected in pleural space 1 week before infection. For the necrostatin (Nec-1s) inhibitor experiment (Biozol, Eching, Germany), 100 µg of Nec-1s was injected in pleural space just before infection. For the zVAD-fmk inhibitor experiment (Invivogen, San Diego, CA, USA), 100 µg of zVAD-fmk was injected in pleural space just before infection. For the pleural RM depletion, 100 µl of clodronate liposome and liposome control (Liposoma B.V.) were injected in pleural space 2 days before infection. For the adoptive transfer of B cells, 106 purified pleural B cells from WT mice in a volume of 100 µl saline was injected in pleural space just before intra-pleural injection of LPS.
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9

Intestinal and Immune Cell Immunomodulation

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For assessing immunomodulation of intestinal cells, HT-29 cells were co-incubated with bacteria at a multiplicity of infection (MOI) of 40 or media, with recombinant TNF-α (Peprotech, London, UK) at a final concentration of 5 ng/mL as previously described (for more details see Supplementary Materials). Supernatants were recovered and stored at −80 °C until subsequent analysis. Interleukin-8 (IL-8) was later quantified in the supernatants using the Human IL-8 ELISA MAX Standard Set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Each strain was tested 3 times from 3 independent cultures.
Immunomodulation of PBMCs was assessed in cells from five healthy donors (for more details, see Supplementary Materials). Bacteria at an MOI of 10 or control media were added for 24 h. Supernatants were recovered and stored at −80 °C until subsequent analysis. IL-10 and IL-12p70 were later quantified in the supernatant by ELISA (Mabtech, Cincinnati, OH, USA) according to the manufacturer’s instructions. Each strain was tested 3 times from 3 independent cultures.
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10

Adipose Tissue Lipolysis Assays

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Adipose tissues were collected and washed in PBS before subjected to lipolysis assays. For isoproterenol stimulation, adipose tissues were cut into small tissue pieces and incubated in serum‐free DMEM–High Glucose (Sigma, D5796) with 2% fatty acid‐free BSA (Sigma, 126579) in the absence or presence of 10 μM isoproterenol (Sigma, I6504) for the indicated time. TNFα‐induced lipolysis was induced as previously described (Ju et al, 2019 (link)). In brief, small adipose tissue pieces were cultured in DMEM–High Glucose for 24 h in the absence or presence of 100 ng/ml recombinant TNFα (Peprotech, 315‐01A) and then transferred into serum‐free DMEM containing 2% fatty acid‐free BSA for 3 h. Supernatants were collected and FFA concentration normalized to adipose tissue input.
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