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Tnf α and il 6

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TNF-α and IL-6 are cytokine proteins that play key roles in the immune response. TNF-α is involved in systemic inflammation, while IL-6 is a pro-inflammatory cytokine. These proteins can be measured in the lab to assess various physiological and pathological conditions.

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Lab products found in correlation

Adiponectin, by Merck Group (2 mentions) Il 1β, by R&D Systems (2 mentions) Synergy ht multi mode reader, by Agilent Technologies (1 mentions) Carrageenan, by Merck Group (1 mentions) Extracellular signal regulated kinase erk, by Cell Signaling Technology (1 mentions) Hsp70, by Santa Cruz Biotechnology (1 mentions) Pge2 assay kit, by R&D Systems (1 mentions) Anti p iκbα, by Cell Signaling Technology (1 mentions) C jun nh2 terminal kinase jnk, by Cell Signaling Technology (1 mentions) Lamin a c, by Santa Cruz Biotechnology (1 mentions) Anti inos, by Cell Signaling Technology (1 mentions) Anti iκbα, by Cell Signaling Technology (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Salmonella minnesota lps, by InvivoGen (1 mentions) Penicillin streptomycin stock solution, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Pam3csk4, by InvivoGen (1 mentions) Mip 2, by R&D Systems (1 mentions) Ifn β, by PBL Assay Science (1 mentions)

18 protocols using tnf α and il 6

1

Inflammatory Biomarker Quantification and Preprocessing

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Three circulating inflammatory markers were derived from serum samples, which were assayed in duplicate using ELISA assay kits [TNF-α and IL-6 from Invitrogen, CRP (high sensitivity) from BioCheck – see Supplementary Table 1 for detection limits and inter- and intra-assay coefficients of variation]. Correlation coefficients were determined by linearity of dilution and were all r ≥ 0.99. IL-6 was skewed (IL-6 skew = 3.97). Because 22 individuals had a 0 for IL-6 (and 0 scores cannot be logged), we first conducted a linear transformation by adding 1 and then natural log-transformed to correct for skewness (n_log IL-6 skew = −0.55).
Thirteen participants had CRP levels of 10 mg/L or higher. Because removal of those with “high CRP” (i.e., greater than 10) is still debated in the literature (Mac Giollabhui et al., 2020 (link)), we conducted analyses on CRP by 1) retaining those with high CRP and correcting for skewness by taking the natural log (raw CRP skew including “high CRP participants” = 2.81; n_log CRP skew including “high CRP participants” = −0.18), and 2) removing those participants with CRP greater than 10 (raw CRP skew without “high CRP participants” = 1.14).
Zuo S., Gibbs E., Kelman Z., Wang T.S., O’Donnell M., MacNeill S.A, & Hurwitz J. (1997). DNA polymerase δ isolated from Schizosaccharomyces pombe contains five subunits. Proceedings of the National Academy of Sciences of the United States of America, 94(21), 11244-11249.
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2

ELISA Quantification of Inflammatory Cytokines

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The concentrations of TNF-α and IL-6 (Invitrogen) in collected supernatants from 24 hr-treated BMDMs were determined using enzyme-linked immunosorbent assay (ELISA) in duplicate following the manufacturer’s instructions. The plates were read on a Synergy HT Multi-Mode Reader (BioTek). Wavelength correction and background signal were subtracted from the absorbance values.
Reverte M., Eren R.O., Jha B., Desponds C., Snäkä T., Prevel F., Isorce N., Lye L.F., Owens K.L., Gazos Lopes U., Beverley S.M, & Fasel N. (2021). The antioxidant response favors Leishmania parasites survival, limits inflammation and reprograms the host cell metabolism. PLoS Pathogens, 17(3), e1009422.
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3

Plasma Markers of ECC-Induced Inflammation

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To measure plasma markers of ECC-induced systemic inflammation in our model, blood samples were obtained at the end of the experiment, and plasma was separated by centrifuging the total sample for 15 min at 1000 ×g and 4°C. The plasma supernatant was removed and stored at −70°C until analysis. Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and neutrophil elastase (NE) in plasma were assayed using the following commercial ELISA kits according to the manufacturers' instructions: TNF-α and IL-6, Thermo Scientific (Rockford, IL, USA); and NE, Cloud-Clone (Houston, TX, USA).
To measure ECC-induced organ damage in our model, the right lung and right kidney were harvested, homogenized, and centrifuged as described [12 (link)]. Briefly, tissues were harvested on ice immediately after euthanasia and weighed. An aliquot of tissue (50 mg) was cut into 1 mm3 pieces, added to 500 μL normal saline, and homogenized. Samples were then left standing on ice for 5 min and centrifuged at 3500 ×g for 20 min at 40°C. Supernatants were transferred to Eppendorf tubes and stored at −70°C until analysis. Samples were assayed for TNF-α, IL-6, and NE as for plasma samples.
Luo S., Tang M., Du L., Gong L., Xu J., Chen Y., Wang Y., Lin K, & An Q. (2015). A Novel Minimal Invasive Mouse Model of Extracorporeal Circulation. Mediators of Inflammation, 2015, 412319.
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4

In Vivo Cytokine Release Analysis

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For analysis of R848-induced in vivo cytokine release, Spopfl/fl and Spopfl/fl-Vav-cre mice were intraperitoneally injected with 50 nmol R848 (in 0.1 ml of PBS) and serum samples were collected 1.5 hours later. Cytokine concentrations of IFN-β, TNF-α, IL-6, and IL-12p40 were determined by ELISA (IFN-β and IL-12p40 from R&D Systems and TNF-α and IL-6 from Thermo Fisher Scientific).
Tawaratsumida K., Redecke V., Wu R., Kuriakose J., Bouchard J.J., Mittag T., Lohman B.K., Mishra A., High A.A, & Häcker H. (2022). A phospho-tyrosine–based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity. Science Advances, 8(27), eabq0084.
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5

In-Vitro Anti-Inflammatory Assay

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Anti-I-κBα, anti-p-I-κBα, anti-iNOS, horseradish peroxidase-conjugated anti-mouse, anti-goat IgGs, and antibody for phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 were purchased from Cell Signaling (Beverely, MA, USA). Anti-anti-NF-κB p65, HSP 70 and Lamin A/C antibodies were supplied from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-murine iNOS was purchased from BD Bioscience (San Jose, CA, USA). PGE2 assay kit was supplied from R&D system (Minneapolis, MN, USA). TNF-α and IL-6 were supplied from Thermo Scientific (Waltham, MA, USA). Polyethylene glycol number 400 (PEG) solution, carrageenan, dexamethasone, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Hwangbo M., Jung J.Y., Ki S.H., Park S.M., Jegal K.H., Cho I.J., Lee J.H., Kang S.H., Park S.D., Ku S.K., Kim S.C., Zhao R.J., Jee S.Y, & Kim Y.W. (2014). U-Bang-Haequi Tang: A Herbal Prescription that Prevents Acute Inflammation through Inhibition of NF-κB-Mediated Inducible Nitric Oxide Synthase. Evidence-based Complementary and Alternative Medicine : eCAM, 2014, 542825.
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6

Cytokine Profiling in Brain Tissue

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Brains were removed and the ipsilateral hemisphere immediately homogenized in cold neuronal protein extraction reagent (ThermoFisher Scientific) using a ratio of 1g of tissue to 10ml of reagent. The samples were centrifuged at 10,000 × g for 20 min at 4°C and the supernatants were used for cytokine measurements. Levels of proinflammatory cytokines were determined by TNFα and IL-6 enzyme-linked immunosorbent assays (ThermoFisher Scientific).
Xu L., Sun X., Griffiths B., Voloboueva L., Valdes A., Dobrenski M., Min J.J, & Stary C.M. (2023). Sexual Dimorphism in Brain Sirtuin-1 and m6A Methylated Sirtuin-1 mRNA, and in Protection with Post-Injury Anti-miR-200c treatment, after Experimental Stroke in Aged Mice. Aging and Disease, 14(3), 892-903.
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7

Cytokine Release Assay for Immune Cells

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Cells were plated in flat-bottomed 24-well plates at a density of 2.5 x 106 cells (for RBC-lysed total bone marrow cells or total splenocytes) in 500 μl of complete cell medium, or in flat-bottomed 96-well plates at a density of 50,000 cells (for HSPC-derived macrophages) in 200 µl of complete cell culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution [Gibco]). Whole blood was diluted 1:2 in complete cell culture medium at a final volume of 200 μl and plated in flat-bottomed 96-well plates. To prevent potential fungal growth, 0.5 μg/ml amphotericin B was added to the cultures of total bone marrow cells, total splenocytes and blood from infected animals. Cells were challenged with 100 ng/ml of Pam3CSK4, 100 ng/ml of ultrapure Salmonella minnesota LPS (all from In vivogen) or 25 x 106 inactivated C. albicans ATCC 26555 yeasts for 24 h, and cell-free supernatants were then harvested and tested for cytokine release using commercial enzyme-linked immunosorbent assay (ELISA) kits [TNF-α and IL-6 (eBioscience)]. Unstimulated cells served as negative controls. Triplicate samples were analyzed in each assay.
Bono C., Guerrero P., Jordán-Pla A., Erades A., Salomonis N., Grimes H.L., Gil M.L, & Yáñez A. (2021). GM-CSF Programs Hematopoietic Stem and Progenitor Cells During Candida albicans Vaccination for Protection Against Reinfection. Frontiers in Immunology, 12, 790309.
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8

Macrophage Cytokine Release Assay

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Macrophages were plated in flat-bottomed 96-well plates at a density of 50,000 cells in 200 μl of complete cell culture medium. Cells were challenged with the indicated stimuli for 24 h, and cell-free supernatants were then harvested and tested for cytokine release using commercial enzyme-linked immunosorbent assay (ELISA) kits (TNF-α and IL-6 [eBioscience]; MIP-2 [R&D Systems]) Unstimulated macrophages served as negative controls. Triplicate samples were analyzed in each assay.
Bono C., Martínez A., Megías J., Gozalbo D., Yáñez A, & Gil M.L. (2020). Dectin-1 Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Trained Macrophages via an Indirect Cell-Autonomous Mechanism. mBio, 11(3), e00781-20.
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9

Modulation of Cytokine Responses in IAV Infection

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IFNβ and cytokine levels were determined in lung homogenates of WT, NOD2-/- and IPS–1-/- mice infected or not with IAV (50 PFU) and treated with LTB4 (1μg/kg) or MDP (10 mg/kg) alone and in combination with LTB4. Lungs were harvested 6 hours following treatments and IFNβ (PBL interferon source, Piscataway, NJ, USA), TNFα and IL–6 (eBioscience, San Diego, CA, USA) levels were measured using specific ELISA kits.
Le Bel M, & Gosselin J. (2015). Leukotriene B4 Enhances NOD2-Dependent Innate Response against Influenza Virus Infection. PLoS ONE, 10(10), e0139856.
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10

Cytokine Profiling in ARDS Model

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The pro-inflammatory cytokines Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), CXCL1 (also known as KC), CXCL2 (also known as Macrophage Inflammatory Protein-2, MIP-2), and Angiopoietin-2 levels were determined in samples by ELISA per the manufacturers' instructions. [TNF-α and IL-6: eBioscience (San Diego, CA); MIP2, KC: R&D Systems (Minneapolis, MN); Angiopoietin 2: Abcam (Cambridge, MA)]. These cytokines were selected for measurement, as they are implicated in the pathophysiology of ARDS in both human and animals as pro-inflammatory (TNF-α, IL-6), or neutrophil-attracting chemokines (KC, MIP-2),15 (link)-19 (link) or they have been recently identified as potential early biomarkers for the clinical syndrome (Angiopoietin-2).26 (link) TNF-α, IL-6, KC, and MIP-2 were measured in the BALF at the time of sacrifice (6 hours), whereas IL-6 and Angiopoietin-2 in plasma at 3 and 6 hours.
Kasotakis G., Galvan M., King E., Sarkar B., Stucchi A., Mizgerd J.P., Burke P.A, & Remick D. (2017). Valproic acid mitigates the inflammatory response and prevents acute respiratory distress syndrome in a murine model of Escherichia coli pneumonia at the expense of bacterial clearance. The journal of trauma and acute care surgery, 82(4), 758-765.
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