Elderberries from 8 genotypes including ‘Bob Gordon’, ‘Dallas’, ‘Ocoee’, ‘Ozone’, ‘Sperandio’, ‘Wyldewood’, ‘York’ (S. nigra subsp. canadensis) and ‘Marge’ (S. nigra subsp. nigra) were harvested at the same location of Mt. Vernon (MO, USA) in 2011. Fruit production details are described in Thomas et al. (2015) . Berries were immediately frozen upon harvest, and later were thawed, de-stemmed, French-pressed, and the juices centrifuged, filtered, lyophilized, and dissolved in DMSO. Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, and phosphate-buffered saline (PBS) were obtained from GIBCO (Gaithersburg, MD, USA). Interferon-γ (IFNγ) was purchased from R & D Systems (Minneapolis, MN, USA). Lipopolysaccharide (LPS) (rough strains) from Escherichia coli F583 (Rd mutant) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA, USA).
Interferon γ ifnγ
Manufactured by R&D Systems
Sourced in United States
Interferon-γ (IFNγ) is a cytokine that plays a crucial role in the immune system. It is produced primarily by activated T cells and natural killer cells and is involved in the regulation of immune responses and inflammatory processes.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Plasmocin, by InvivoGen (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps rough strains from escherichia coli f583 rd mutant, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Dmem f12, by PAN Biotech (2 mentions)
Tumor necrosis factor α tnf α, by R&D Systems (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Easysep direct human neutrophil isolation kit, by STEMCELL (1 mentions)
Fstl1, by R&D Systems (1 mentions)
Interleukin 4 il 4, by R&D Systems (1 mentions)
Interleukin 13 il 13, by R&D Systems (1 mentions)
Leukotriene c4 ltc4, by Cayman Chemical (1 mentions)
E selectin, by Thermo Fisher Scientific (1 mentions)
20 protocols using interferon γ ifnγ
1
Elderberry Genotypes Comparison
2
Neutrophil Isolation and Stimulation
3
Macrolide Effects on Septic Endothelial Cells
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ECs were exposed in vitro to septic stimulation for 24 h, by an association of proinflammatory cytokines increased during sepsis: Interferon γ (IFN‐γ) 15 ng/ml (R&D Systems), TNF‐α 5 ng/ml (Peprotech) and lipopolysaccharide (LPS) 100 ng/ml extracted from Escherichia coli. After 24 h of septic stimulation, ECs were incubated with macrolides: Spiramycin, Erythromycin, or Clarithromycin (Merck) at the indicated concentrations for another 24 h. Parallel cultures were carried out with the relevant diluent before phenotypic, quantitative polymerase chain reaction (qPCR), and functional assays.
4
Polarization and Differentiation of Immune Cells
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All cells were maintained in appropriate media for each cell-line recommended by ATCC supplemented with 10% New Born Calf serum (NBCS) (Biofluids, Rockville, MD), penicillin (10 U/ml) and streptomycin (10 μg/ml, Biofluids) at 37°C in a humidified chamber with 5% CO2 atmosphere. Experiments with PA were carried out by treating the cells with indicated concentrations of PA dissolved in appropriate media with 0.1% NBCS. For polarization to M1 type mФ, ANA-1 cells were exposed to 10 ng/ml interferon-γ (IFNγ) for 8 hours (R&D Systems, Minneapolis, MN). For differentiation of U-937 monocytes into mФ, cells were treated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma; P1585) for 24 hours. After replacing with fresh media containing no PMA, the cells were allowed to grow for 48 hours before treatment with PA. CD4+CD25- cells were isolated from the spleens of C57BL/6 mice as previously described [20 (link)]. Briefly, the mФ and B cells were depleted before isolation of CD4+CD25- T cells using MACS separator along with CD4 and CD25 microbeads (Miltenyi Biotec, Auburn, CA).
5
Synthesis and Characterization of Ilantide Peptides
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The Ilantide peptide (SGRKSSKMQA), scrambled peptide1 (KQSAGKRSMS), scrambled peptide2 (KASQKGMSRS) and Ilantide peptide with a reversed sequence (AQMKSSKRGS) were purchased from Schafer-N (Copenhagen, Denmark). The peptides were synthesized using the fluorenylmethyloxycarbonyl protection strategy on TentaGel resin (Rapp Polymere, Tübingen, Germany). The peptides were synthesized as either monomers (Ilantide-m) or dendrimers composed of four monomers coupled to a lysine backbone (Ilantide-t) and were further purified by gel filtration using Sephadex G-10 (Amersham Biosciences, Uppsala, Sweden). The peptides were at least 85% pure according to estimation by high-performance liquid chromatography. The recombinant proteins (that is, IL-1β, IL-6, TNF-α, interferon γ (IFN-γ), IL-1Ra and the ectodomain of IL-1RI) were purchased from R&D Systems (Minneapolis, MN, USA). Ana (Kineret) was obtained from Amgen (Thousand Oaks, CA, USA).
6
Isolation and Culture of Human ASCs
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Specimens of subcutaneous abdominal fat were taken from the patients by 18 G needle biopsy. Tissue processing, ASCs isolation, and culture were performed as described previously (Skalska and Kontny, 2012 (link)). Five human adipose-derived mesenchymal cell lines from healthy volunteers (Lonza Group, Lonza Walkershille Inc., MD, United States; donor numbers: 0000440549, 0000410252, 0000535975, 0000605220, 0000550179) were used as a control. All experiments were performed using ASCs at 3–5 passages. ASCs were cultured in a complete culture medium composed of DMEM/F12 (PAN Biotech United Kingdom Ltd., Wimborn, United Kingdom), 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 200 U/ml penicillin, 200 μg/ml streptomycin (Polfa Tarchomin S.A., Warsaw, Poland), and 5 μg/ml plasmocin (InvivoGen, San Diego, CA, United States). Both untreated and cytokine-licensed ASCs were applied. To this aim, ASCs were stimulated for 24 h with human recombinant tumor necrosis factor (TNF) and interferon γ (IFNγ) (both from R&D Systems, Minneapolis, MN, United States; each applied at 10 ng/ml) (ASCsTI).
7
Dose-response curves of immune cell stimulation
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Dose–response and timing curves were performed for each stimulus. Ficoll-separated PBMCs (4 × 106 cells/well) and MDMs (5 × 105 cells/well) were stimulated for 24 h with LPS (50 µg/mL), peptidoglycan (PGN) (10 µg/mL), lipoteichoic acid (LTA) (1 µg/mL), ssRNA40, a uridine-rich ssRNA analog of HIV-1 ssRNA (6,25 µg/mL) (InvivoGen, San Diego, CA, USA), interferon-γ (IFNγ) (100 U/mL), anti-CD28 (1.25 µg/mL) (R&D System, Minneapolis, MN, USA), and anti-CD3 (2.5 µg/mL) (BD Pharmigen, San Diego, CA, USA). After 24 h, the cells were harvested for flow cytometry analyses; the supernatants of a subset of 5 HIV−, 5 HIV+ untreated, and 11 HIV + cART (5 INRs and 6 FRs) patients were collected and stored for the Luminex assay.
8
Immune Cell Phenotyping and Cytokine Profiling
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Cell staining was performed using fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or pacific blue (PB)-conjugated mouse monoclonal antibodies against CD80, CD86, CD83, CD14, and CD163 (all purchased from BD Biosciences, San Jose, CA, USA). Fluorochrome conjugated antibodies against CD8, CD4, CD56, and CD19 were purchased from BioLegends (Nordic BioSite AS, Kristiansand, Norway). The following cytokines were used: Interleukin-4 (IL-4), granulocyte-colony stimulating factor (GM-CSF), monocyte-colony stimulating factor (M-CSF), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and interferon-γ (IFN-γ; all purchased from R&D Systems (Minneapolis, MN, USA).
9
Evaluating Anti-inflammatory Effects of MSCs
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To evaluate the anti-inflammatory effect of MSCs-CM and MSCs, we measured the serum concentration of the following cytokines by enzyme-linked immunosorbent assay (ELISA) on day 3 after induction of ALF: Granulocyte colony-stimulating factor (G-CSF)/CSF-3, IL-6, eotaxin/chemokine CC ligand (CCL) 11, IL-1α, IL-1β, IL-10, IL-2, TNF-α, and chemokine CXC ligand (CXCL) 1 (R & D Systems).
To evaluate whether STAT3 signaling pathway inhibitor neutralized the anti-inflammatory effect of IL-10, we injected AG490 after infusion of IL-10 and then measured the serum concentration of the following cytokines by ELISA: TNF-α, interferon-γ (IFN-γ), and IL-6 (R & D Systems).
To evaluate whether STAT3 signaling pathway inhibitor neutralized the anti-inflammatory effect of IL-10, we injected AG490 after infusion of IL-10 and then measured the serum concentration of the following cytokines by ELISA: TNF-α, interferon-γ (IFN-γ), and IL-6 (R & D Systems).
10
Isolation and Culture of ASCs from SLE and SSc Patients
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Subcutaneous abdominal fat tissue procurement from SLE or SSc patients, tissue processing, and isolation of ASCs was performed according to the previously described protocol [81 (link)]. Five earlier characterised [46 (link)], human ASCs lines (Lonza Group, Lonza Walkershille Inc., Walkersville, MD, USA) were used as a reference. All experiments were performed using ASCs at 3–5 passages. ASCs were cultured in a complete culture medium composed of DMEM/F12 (PAN Biotech UK Ltd., Wimborne, UK), 10% foetal calf serum (FCS) (Biochrom, Berlin, Germany), 200 U/mL penicillin, 200 µg/mL streptomycin (Polfa Tarchomin S.A., Warsaw, Poland), and 5 µg/mL plasmocin (InvivoGen, San Diego, CA, USA). In addition, for some experiments, ASCs were stimulated for 24 h with human recombinant tumor necrosis factor (TNF) and interferon γ (IFNγ) (both from R&D Systems, Minneapolis, MN, USA; each applied at 10 ng/mL).
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