PBL transmigration was assessed using a 12-well format as previously described (24 (link)). First passage HUVECs were seeded onto 12-well tissue culture plates at a density to yield a confluent monolayer within 24 hours. The HUVECs were stimulated with 100 U/ml tumour necrosis factor-α (TNF-α) (R&D Systems) and 10 ng/ml interferon-γ (IFN-γ) (PreproTech) for 24 hours prior to carrying out adhesion assays with lymphocytes. HUVECs were washed with M199 0.15% BSA to remove residual cytokines and purified PBLs were added. The PBLs were allowed to settle, adhere and transmigrate through the HUVEC monolayer at 37 °C in a CO2 incubator for 7 minutes, a time point previously shown to yield a sufficient proportion of migrated PBLs (25 (link)). Non-adherent cells were removed from the HUVECs by gently washing with M199 0.15% BSA, and video recordings of five fields of view of the endothelial monolayer were made using phase-contrast video microscopy. The video recordings were analysed using Image-Pro Plus software (DataCell). Each PBL was classified as either phase bright and adherent to the surface of the HUVEC monolayer, or phase dark with altered morphology and migrating below the HUVEC monolayer. The percentage of adherent PBL that had transmigrated was calculated.
Interferon γ ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States
Interferon-γ (IFN-γ) is a cytokine that plays a crucial role in the immune system. It is produced by various cell types, including T cells and natural killer cells, and is involved in the regulation of immune and inflammatory responses. IFN-γ has a core function of activating macrophages, enhancing antigen presentation, and promoting the differentiation of Th1 cells, which are important for cell-mediated immunity.
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Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharide lps, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Tumor necrosis factor α tnf α, by Thermo Fisher Scientific (3 mentions)
Tnf α, by R&D Systems (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium f12, by Merck Group (2 mentions)
04 001 1acs, by Sartorius (2 mentions)
Penicillin streptomycin and l glutamine, by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (2 mentions)
Mouse colony stimulating factor 1 csf1, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Rabbit anti iba1 antibody, by Fujifilm (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
31 protocols using interferon γ ifn γ
1
Quantifying Lymphocyte Transmigration Across Activated Endothelium
2
Microglia Cell Culture Reagents and Protocols
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Cell culture reagents including Hank's Balanced Saline Solution (HBSS), 10,000 U/ml Penicillin/10,000 μg/ml streptomycin (PS), fetal bovine serum (FBS), horse serum, Dulbecco's Modified Eagle Medium with Ham's F-12 supplement (DMEM/F12), and 0.25% (w/v) Trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) were from Gibco (Thermo-Fisher Scientific, Burlington, ON, Canada). Rabbit anti-Iba1 antibody was from Wako (Osaka, Japan; RRID:AB_839504 ). Reagents for fluorescence assays including 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a, 4a-Diaza-s-Indacene (BODIPY 493/503), To-Pro-3 iodide, and donkey anti-rabbit Alexa Fluor 488 antibodies (RRID:AB_2535792 ) were from Invitrogen (Thermo Fisher Scientific, Burlington, ON, Canada). Chemicals and reagents were from Millipore Sigma (St. Louis, MO, USA) including Escherichia coli O111:B4 lipopolysaccharide (LPS), poly-l -lysine (PLL), and all SCCA used in culture experiments. Interferon γ (IFNγ) was from Peprotech (Montreal, QC, Canada). Enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Toronto, ON, Canada). Phosphate buffered saline was prepared from tablets to pH 7.4 (Bioshop Canada, Burlington, ON, Canada).
3
Th1 Cell Differentiation Protocol
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CD4+ CD62Lhi CD25− B220− T cells were isolated by cell sorter (MoFlo; Beckman Coulter, Brea, CA) from 5‐week‐old BDC2.5 TCR transgenic NOD mice and differentiated into Th1 cells by culturing them in Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, 50 μm β‐mercaptoethanol, l ‐glutamine (2 mm ) and 50 U/ml penicillin and streptomycin (Gibco) with plate‐bound anti‐CD3 (2 μg/ml, clone 145‐2C11, grown in‐house), soluble anti‐CD28 (10 μg/ml; BD Pharmingen, Franklin Lakes, NJ) and interleukin‐2 (IL‐2; 100 U/ml), IL‐12 (10 ng/ml) and interferon‐γ (IFN‐γ; 100 U/ml) (all from Peprotech, Rocky Hill, NJ) for 4 days at 37° with 5% CO2. Afterwards, the production of IFN‐γ was checked by specific ELISA (R&D Systems, Minneapolis, MN) and/or intracellular staining.
4
Culturing Human Leukemia and Carcinoma Cells
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The human acute T lymphocytic leukemia cell line (Jurkat T cells) and the human tongue squamous cell carcinoma cell line (Cal27 cells) were purchased from the cell bank of the typical Culture Preservation Committee of the Chinese Academy of Science (Shanghai, China). The Jurkat cells were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (04-001-1ACS, Biological Industries, Kibbutz Beit-Haemek, Israel). The Cal27 cells were grown in Dulbecco’s modified Eagle’s medium/F12 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (10099-141, Gibco, Grand Island, NY). PBMCs were isolated from peripheral blood donated by healthy adults through density gradient centrifugation and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (10099-141, Gibco), interferon γ (IFN-γ; 300-02, 2000 U/mL, PeproTech, Rocky Hill, NJ), and IL-2 (200-02, 10 ng/mL, PeproTech).
5
Immunophenotyping of Lymphocyte and Hepatocyte Subsets
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For analysis of early stimulation on day 3, unlabeled PBMCs were retrieved from culture and stained with monoclonal antibodies as indicated: fluorescein isothiocyanate–conjugated CD4 and CD8 (Immuno Tools, Friesoythe, Germany), phycoerythrin‐conjugated CD25 and CD69 (BD Bioscience, San Jose, CA). For late proliferation control, PKH‐26–stained PBMCs were analyzed on day 10. Additional staining for CD4 and CD8 was performed to distinguish T cell subpopulations. Expanded Tregs were excluded during gating for analysis by CellVue‐labeling (Supporting Fig. 1) to prevent confounding. Flow cytometric measurements were performed using a FACSCalibur (BD Biosciences), and results were analyzed by FACSDiva software.
Hepatocytes retrieved from MLHCs on day 10 were stained with allophycocyanin‐conjugated anti–HLA‐DR for detection of major histocompatibility complex (MHC) class II expression. MHC class II expression also was examined on PHH monocultures stimulated with interferon γ (IFNγ) (100 ng/mL; Peprotech, Rocky Hill, NJ) for 3 days, with nonstimulated cells serving as control. Flow cytometric analyses were performed using a FACSCanto II (BD Biosciences).
Hepatocytes retrieved from MLHCs on day 10 were stained with allophycocyanin‐conjugated anti–HLA‐DR for detection of major histocompatibility complex (MHC) class II expression. MHC class II expression also was examined on PHH monocultures stimulated with interferon γ (IFNγ) (100 ng/mL; Peprotech, Rocky Hill, NJ) for 3 days, with nonstimulated cells serving as control. Flow cytometric analyses were performed using a FACSCanto II (BD Biosciences).
6
Murine Bone Marrow Macrophage and Dendritic Cell Culture
7
Expansion of Cytokine-Induced Killer Cells
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The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Human Research Ethics committee of Lanzhou University Second Hospital (ethics approval number: 2017A-044). Informed consent was obtained from all individual participants included in the study. PBMCs were obtained using Ficoll-Hypaque density centrifugation. Cells were resuspended at 1×106 CFU/mL in X-VIVO-20 media (Lonza, Basel, Switzerland) with 1,000 U/mL interferon-γ (IFN-γ, Peprotech, Rocky Hill, NJ, USA) for the first 24 h. On day 1, 50 ng/mL anti-CD3 monoclonal antibody (OKT3, Peprotech, Rocky Hill, NJ, USA), 100 U/mL interleukin-1α (IL-1α, Peprotech, Rocky Hill, NJ, USA) and IL-2 (Peprotech, Rocky Hill, NJ, USA) were added to the medium. Cells were cultured in 5% CO2 at 37 °C, and fresh medium and IL-2 were added every two days. The concentration of IL-2 was 300, 500, and 1,000 U/mL in the first week; and 300, 500, 1,000, 3,000, and 6,000 U/mL in the second week, respectively. CIKs were harvested on day 16.
8
Generating M1 Macrophages from RAW264.7 and Peritoneal Cells
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293T cells and RAW264.7 cells of the mouse macrophage cell line were obtained from the Cell Bank of Chinese Academy of Sciences. Peritoneal macrophage (PM) was derived from peritoneal dropsy of female C57BL/6 mice (8-10 weeks old) according to previous study 30 (link). In brief, mice were injected with 2 ml 3% fluid thioglycolate medium brewer modified (BD, Franklin Lakes, NJ, USA) to elicit peritoneal macrophage. After 4 days, the mice were sacrificed, and the peritoneal cavity of mice were injected with 5ml pre-chilled 1×PBS buffer followed by massage for 5min. Peritoneal macrophage were immediately collected. All cells were cultured in DMEM (Bioind, Kibbuiz, Israel) containing 10% FBS (Bioind, Kibbuiz, Israel) and 1% penicillin/ streptomycin. The cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.
To generate M1 macrophages, RAW264.7 cells and PM were stimulated with 100ng/ml lipopolysaccharides (LPS) (Sigma-Aldrich, St. Louis, MO, USA) and 20ng/ml interferon-γ (IFNγ) (Peprotech, Chicago, IL, USA) for 24h.
To generate M1 macrophages, RAW264.7 cells and PM were stimulated with 100ng/ml lipopolysaccharides (LPS) (Sigma-Aldrich, St. Louis, MO, USA) and 20ng/ml interferon-γ (IFNγ) (Peprotech, Chicago, IL, USA) for 24h.
9
Cell Culturing HK2 Proximal Tubular Cells
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Human kidney 2 (HK2) proximal tubular cell line (ATCC Cat# CRL-2190, RRID:CVCL_0302; Manassas, VA, USA) was maintained in RPMI supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA), 1% insulin–transferrin–selenium (Thermo Fisher Scientific) and 36 ng/mL hydrocortisone (Sigma-Aldrich). Cells were made quiescent by overnight incubation in serum-free RPMI and then pretreated for 90 min with hidrosmin (range of doses 0.1–1 mM) or vehicle of the highest dose (0.5% DMSO) before stimulation with either high-glucose (30 mM D-Glucose; Sigma-Aldrich) for 24 h, or a combination of human cytokines interleukin-6 (IL-6, 102 U/mL) and interferon-γ (IFNγ, 103 U/mL) (PeproTech, Rocky Hill, NJ, USA) for 6 and 24 h.
10
Murine Bone Marrow-Derived Macrophage Isolation
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Bone marrow-derived macrophages were obtained from male and female C57BL/6 wildtype mice (5–7 months old), which were received from the animal facility of the University Hospital Frankfurt. The mice were euthanized and both legs together with the hip were extracted. Tibias, femurs and the hip were individually flushed with sterile PBS, the cell suspension was collected, centrifuged and the pellet resuspended in ACK buffer (Lonza) to remove erythrocytes. The obtained white pellet was resuspended in DMEM containing 20 ng/ml macrophage colony-stimulating factor (M-CSF) (Biotrend) and 4×106 cells per well were seeded on a 6 well plate. After 7 days of differentiation macrophages were activated overnight in DMEM containing 25 ng/ml interferon-γ (IFN-γ) (PeProtech) and 100 ng/ml lipopolysaccharide (LPS) (Enzo Life Science) for M1 cells; and 25 ng/ml interleukin-4 (PeProTech) for M2 cells. DMEM was supplemented with 10% fetal calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).
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