PN and PH cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEMZF12) medium supplemented with 3% FBS and γ-interferon (5 U/ml; Sigma-Aldrich) at 33°C and 5% CO2 and plated in a 6-well plate for 24 h. The cells were then changed into γ-interferon-free medium and maintained at 37°C for 4 days before being used in the experiment. cAMP levels were measured with a direct cAMP Enzyme Immunoassay Kit (Sigma-Aldrich, #CA200) according to the manufacturer’s protocol. The results are expressed as pmole/ml. Statistical analysis was performed using a two-tailed Student’s t-test.
Interferon γ
Manufactured by Merck Group
Sourced in United States
Interferon-γ is a cytokine produced by immune cells, such as T cells and natural killer cells, that plays a crucial role in the body's immune response. It is commonly used in research and diagnostic applications to study and evaluate immune system function.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharide, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Foetal calf serum, by Lonza (2 mentions)
Il 1β, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Type 1 collagen, by BD (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Direct camp enzyme immunoassay kit, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Mouse recombinant interferon γ, by R&D Systems (1 mentions)
Vi cell xr, by Beckman Coulter (1 mentions)
Gen5 2, by Agilent Technologies (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
28 protocols using interferon γ
1
cAMP levels in PN and PH cells
2
Cell culture viability and growth
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All cells were cultured under identical conditions, including cell density and passage number, and studied at ~80% confluency on 100 mm plastic culture plates in complete medium. Indicated cultures included rapamycin (Sigma, St Louis, MO, USA) or mouse recombinant interferon-γ (R&D Systems, Minneapolis, MN, USA) for 48 h (B16 and ID8agg) or 60 h (ES2) at concentrations shown. Cell viability and numbers were determined on a Vi-Cell XR (Beckman Coulter, Brea, CA, USA) or hemocytometer and stained as described below. Dimethylsulfoxide (Sigma) or phosphate-buffered saline was used as negative controls (vehicle) for rapamycin or interferon-γ, respectively.
3
Quantifying Nitric Oxide Production by Activated Macrophages
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Nitric oxide (NO) synthesized by activated Mφ was quantitated by measuring the level of the nitrite ion (NO2−) in the incubation medium (Nowosielska et al. 2011 (link)). Mφ were suspended in CM supplemented with 50 U/ml interferon-γ (IFN-γ; Sigma, Poznan, Poland) and 100 ng/ml lipopolysaccharide (LPS; Sigma) and incubated for 48 h in SC; for each experimental group eight samples were used. After that, 100 µl of the supernatant was mixed with 100 µl of the Griess reagent and kept in the dark for 10 min at RT. Absorbance at 540 nm was then measured using the microplate spectrophotometer Epoch™ (BioTek® Instruments, Inc., Vermont, USA). The obtained data were analyzed with use of the reader software Gen5™ 2.0 (BioTek® Instruments, Inc., Vermont, USA).
4
Culturing Human and Murine Cell Lines
5
Immortalized Mouse Podocyte Transfection
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We use a well-described and characterized immortalized mouse podocyte cell line (33 (link)). Cells were maintained and differentiated as previously described (26 (link)) with the following modifications. The culture medium was glucose-free RPMI-1640 supplemented with 5.5 mM d -glucose, 10% FBS, 10 µg/ml penicillin, 10 µg/ml streptomycin. For undifferentiated cells, 10 U/ml interferon-γ (Sigma-Aldrich) was used. Cells were differentiated for 7–14 days. Differentiated immortalized podocytes were transiently transfected with SGLT2-ires-CFP (GenScript, Piscataway, NJ) or empty vector CFP (Addgene, Cambridge, MA). DNA plasmids were delivered to the cells using Lipofectamine LTX reagent with plus reagent (ThermoFisher) diluted in Opti-MEM (ThermoFisher) according to the manufacturer’s instructions. The final DNA concentration in each well was 500 ng/ml. Cells were transfected for 48 h and characterized with SGLT2-ires-CFP fluorescence and anti-SGLT2 antibodies.
6
Stimulation of Cultured Cells with TLR Agonists
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Cultured cells were treated with different concentrations of TLR agonists for 24 hours in the presence of interferon-γ (100 U/ml; Sigma, Taufkirchen, Germany). Tripalmitoyl-S-glyceryl-cysteine (Pam3CSK4; EMC Microcollections GmbH, Tübingen, Germany) was used as specific agonist of TLR1/2. For activation of TLR4, microglial cells were exposed to endotoxin (LPS) from Escherichia coli serotype 026:B6 (Sigma, Taufkirchen, Germany). CpG oligodesoxynucleotide (ODN) 1668 (TCC ATG ACG TTC CTG ATG CT) from TIB Molbiol (Berlin, Germany) was used as specific ligand of TLR9. Unstimulated cells were treated with cell culture medium containing 100 U/ml interferon-γ. After 24 hours of stimulation, supernatants were stored at –80°C until measurement of NO, cytokine and chemokine levels. Cells were used in the bacterial phagocytosis assay. Cell viability was determined using the WST-1 Cell Proliferation Reagent (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.
7
Cytokine and TLR Agonist Stimulation in Transwell
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Cytokines were added to the basal Transwell chamber at the following final concentrations: Tumour Necrosis Factor-α (1 ng/ml, 10 ng/ml, 100 ng/ml, Sigma, Saint Louis, USA), Interferon-γ (1 ng/ml, 10 ng/ml, 100 ng/ml, Sigma, Saint Louis, USA), IL-1β (1 ng/ml, 5 ng/ml, 10 ng/ml Sigma, Saint Louis, USA) IL-17A (50 ng/ml, 100 ng/ml, Gibco, Life Technology, USA), IL-22 (50 ng/ml, 100 ng/ml, Sigma, Saint Louis, USA), and IL-26 (50 ng/ml, 100 ng/ml Abnova Taiwan Corp). TLR agonists were added to the apical and basal Transwell chambers at the following final concentrations: TLR1: Pam3CSK4 (1 µg/ml), TLR2: HKLM (108 cells/ml), TLR3: Poly(I:C) HMW (10 µg/ml), TLR3: Poly (I:C) LMW (10 µg/ml), TLR4: LPS (1 µg/ml), TLR5: Flagellin (1 µg/ml), TLR6: FSL-1 (1 µg/ml), TLR7: Imiquimod (1 µg/ml), TLR8: ssRNA40 (1 µg/ml), TLR9: ODN2006 (5 µM).
8
Culturing Auditory Cell Lines for Viability Assays
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The auditory OC-k3 and HEI-OC1 cell lines were cultured according to Kalinec et al.82 , in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 2 mM glutamine and 10% fetal bovine serum (Gibco, Grand Island, NY, USA), and in the absence of antibiotics. The cells were passaged at a density of 2 × 105 cells/mL and maintained under permissive conditions (i.e., 33 °C and 10% CO2, with addition of 50 U/mL interferon-γ (Sigma-Aldrich, St. Louis, MO, USA) to OC-k3 cultures).
When conducting MTT (Sigma Aldrich (St. Louis, MO, USA)) and ATP-based (Promega Corp, Madison, WI, USA) viability assays, the cells were plated onto 96-well flat-bottom plates (100 μL cell suspension/well) 24 h prior to AG treatment. Viability measurements were carried out following the manufacturer instruction.
When conducting MTT (Sigma Aldrich (St. Louis, MO, USA)) and ATP-based (Promega Corp, Madison, WI, USA) viability assays, the cells were plated onto 96-well flat-bottom plates (100 μL cell suspension/well) 24 h prior to AG treatment. Viability measurements were carried out following the manufacturer instruction.
9
Culturing Human and Murine Cell Lines
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K562 cells (provided by M. E. Hemler, Dana-Farber Cancer Institute, Boston, MA, USA) were cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal calf serum (FCS; Lonza Bioscience) and 2 mM L-glutamine. Telomerase-immortalised HFF (provided by K. Clark, University of Leicester, Leicester, UK), conditionally immortalised MEF (generated in-house, see ref. 57 (link)), A375-SM (provided by I. J. Fidler, MD Anderson Cancer Center, Houston, TX, USA) and osteosarcoma (U2OS; purchased from Sigma Aldrich, 92022711) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FCS and 2 mM L-glutamine. MEF cells were supplemented with interferon-γ (Sigma-Aldrich). All cells were maintained at 37 °C in a humidified 5% (v/v) CO2 atmosphere, except for MEF cells, which were maintained at 33 °C. All cell lines were frequently tested for mycoplasma and were negative. Cell lines were not authenticated and are not listed in the database of commonly misidentified cell lines maintained by ICLAC (http://iclac.org ) and NCBI Biosample (http://www.ncbi.nlm.nih.gov/biosample ).
10
Culturing HEK293 and MV^D7 cells
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HEK293 cells were obtained from Simon Rothenfußer (Klinikum der Universitat München, München, Germany) as previously described (Menon et al., 2015 (link)). Cells were cultured in DMEM (Gibco cat. no. 11965092) with 10% FBS (Hyclone) and maintained at 5% CO2 at 37°C. MVD7 cells (Bear et al., 2000 (link)) were a gift from Frank Gertler (MIT, MA, USA). Cells were maintained in DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, cat. no. 35050061), 15% FBS (Thermo Fisher cat. no. FB12999102), and 50 U/mL of interferon-γ (Sigma cat. no. IF005). Cells were cultured at 32°C with 5% CO2. Cells lines are routinely tested for Mycoplasma by qPCR.
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