Splenocytes (1 × 106) from mixed WT and dKO BM chimeric mice were incubated in 96-well tissue culture plates coated with anti-NK1.1 (PK136) or control mouse IgG2a (39 (link)). For cytokine response testing, 1 × 106 splenocytes from mixed WT and dKO BM chimeric mice were incubated with 2.5 ng/ml mouse IL-12 and 2.5 ng/ml mouse IL-18 (R&D Systems). To test degranulation, 1 × 106 splenocytes from mixed WT and dKO BM chimeric mice were co-cultured with either 1 × 105 untransfected or m157-transfected B6 3T3 cells for 5 h at 37° C with PE-conjugated anti-CD107a (clone 1D4B) and GolgiStop (BD Biosciences), followed by staining for surface molecules and intracellular IFN-γ. For assessment of phosphorylated STAT4 and STAT1, 1–3 × 106 splenocytes were cultured with 20 ng/ml mouse IL-12 for 30 min or 1000 U/ml mouse IFN-α (PBL Assay Science) for 10 min, fixed, and stained with AlexaFluor 647-conjugated phosphorylated STAT4 (pY693, clone 38/p-Stat4) or PE-conjugated phosphorylated STAT1 (pY701, clone 4a) (BD Biosciences) (40 (link)).
Mouse ifn α
Manufactured by PBL Assay Science
Sourced in United States
The Mouse IFN-α is a laboratory equipment used to measure the levels of interferon-alpha in mouse samples. It is a tool for researchers to quantify this important immune system protein.
Automatically generated - may contain errors
Lab products found in correlation
Mouse il 12, by R&D Systems (1 mentions)
Mouse il 18, by R&D Systems (1 mentions)
Golgistop, by BD (1 mentions)
Ruxolitinib, by Cell Guidance Systems (1 mentions)
Cell culture media and supplements, by Thermo Fisher Scientific (1 mentions)
Mouse ifn γ, by Merck Group (1 mentions)
Human ifn β, by PBL Assay Science (1 mentions)
Human il 6, by BD (1 mentions)
Mouse ifn β, by PBL Assay Science (1 mentions)
Mouse il 6, by BD (1 mentions)
Mouse tnf α, by BD (1 mentions)
24 well dishes, by Corning (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Human ifn γ, by R&D Systems (1 mentions)
Mouse ifn β, by R&D Systems (1 mentions)
Human ifn β, by R&D Systems (1 mentions)
Mouse ifn γ, by R&D Systems (1 mentions)
Human ifnα, by PBL Assay Science (1 mentions)
Captopril, by Merck Group (1 mentions)
Lys des arg9 bradykinin, by Merck Group (1 mentions)
6 protocols using mouse ifn α
1
Cytokine and Degranulation Assays for NK Cells
2
Mouse Fibroblast Isolation and Immortalization
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Primary mouse fibroblasts (mouse embryonic fibroblasts [MEF] and mouse newborn cells [MNC]) were isolated from C57BL/6 and BALB/c mouse embryos and newborns according to described protocols (65 (link)). Immortalized mouse fibroblasts were generated from primary C57BL/6 and BALB/c MEF by crisis immortalization (66 (link)). HeLa:IFNLR and 3T3-ISREluc:IFNLR cells were generated by transfection of HeLa cells (ATCC CCL-2) and NIH 3T3:ISREluc cells (11 (link)) with plasmids pcDNA-hIFNLR1 (subcloned from pUNO1-hIL28R; InvivoGen) and pUNO1-mIL28R (InvivoGen), respectively, and subsequent selection with the appropriate agent. All cells were grown in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamine. Cell culture media and supplements were obtained from Gibco/Life Technologies. Mouse IFN-α was purchased from PBL, mouse IFN-γ was purchased from Merck Millipore, human and mouse IFN-λ was purchased from R&D Systems, and ruxolitinib was purchased from Cell Guidance Systems.
3
Cytokine Production Analysis by ELISA
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ELISA was used to detect the production of pro-inflammatory cytokines and type I interferon from cells. After infection, treatment and transfection of stimulants, cell supernatant was collected and analyzed cytokine production levels. Mouse IFN-α (PBL interferon source), mouse IFN-β (PBL interferon source), mouse IL-6 (BD biosciences) and mouse TNF-α (BD biosciences), human IFN-β (PBL interferon source) and human IL-6 (BD biosciences) were used for analysis according to manufacturer’s protocol.
4
Mouse Macrophage IFN Response Assay
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RAW264.7 cells, a mouse macrophage-like cell line purchased from the ATCC, were maintained in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% FBS and 1% penicillin/streptomycin.
For type and type II IFN treatment, 1×105 RAW264.7 cells were plated in 24-well dishes (Costar), and the plates were incubated at 37 °C. After 24 h, 10 or 102 ng/mL mouse IFN-α (PBL Assay Science), mouse IFN-β (PBS Assay Science), and IFN-γ (Peprotech) were added to the appropriate wells. After 24 h, the culture supernatants and RAW264.7 cells were harvested for ELISA and qRT-PCR analysis, respectively.
For type and type II IFN treatment, 1×105 RAW264.7 cells were plated in 24-well dishes (Costar), and the plates were incubated at 37 °C. After 24 h, 10 or 102 ng/mL mouse IFN-α (PBL Assay Science), mouse IFN-β (PBS Assay Science), and IFN-γ (Peprotech) were added to the appropriate wells. After 24 h, the culture supernatants and RAW264.7 cells were harvested for ELISA and qRT-PCR analysis, respectively.
5
Quantifying Cytokine Levels by ELISA
6
Immune Cell Stimulation Assay
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The following reagents were used to stimulate cells: Recombinant human IFN-α (1,000 U/ml, PBL Assay Science, Piscataway, NJ, USA), mouse IFN-α (1,000 U/ml, PBL Assay Science), CpG-B 1826 (10 µg/ml), CpG-A-2336 (5 µg/ml) (IDT Biotechnologies, Coralville, IA, USA), CpG-A-1585 (1 µg/ml) (Invivogen, San Diego, CA, USA), resiquimod (1 µg/ml) (R848; Invivogen) (23 (link), 25 (link)–27 (link)), bradykinin peptide (10 µM), Lys-des-Arg(9)-bradykinin, which is a kinin breakdown product and a selective bradykinin B1 receptor agonist (10 µM), Arg–Pro–Hyp–Gly–Phe–Ser–Pro–Phe–Arg B2 receptor agonist (10 µM), B1 receptor antagonist ([des-Arg10-HOE140]- DH-1 μg/ml), B2 receptor antagonist (HOE140–H–10 μM) (all bradykinin agonists and antagonists were purchased from Sigma-Aldrich, St. Louis, MO, USA) (28 (link)), recombinant human klk1 (1 µg/ml) (Creative Biomart, Shirley, NY, USA), captopril (20 μM)(Sigma) (29 (link)), and indomethacin (indo) (1 µg/ml) (Sigma) (30 (link)).
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