Murine Peritoneal Macrophage cells were seeded in 12-well plate (Thermo Scientific) at density of 1 × 106 per well in 1 mL RPMI 1640 [supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL)] and incubated overnight. Peritoneal macrophage was treated with medium, Pam3CSK4 (InvivoGen, 100 ng/mL) plus murine IFN-γ (40 ng/mL, Peprotech), LPS (InvivoGen, 100 ng/mL) plus murine IFN-γ (40 ng/mL, Peprotech) and indicated SMU-Y6 for 24 h. Cells were collected into centrifuge tube after trypsinization and carefully aspirated the supernatant after centrifugation at 300×g for 5 min. The cells were washed twice with PBS and stained with a mixture of antibodies for 30 min, including anti-mouse CD86-PE-Cy7 (BD Biosciences), anti-mouse CD11b-FITC (BD Biosciences). After incubation, the cells were centrifuged at 300×g for 5 min and washed twice with PBS. Stained cells were analyzed by flow cytometer (FACScanto II, BD) and the flow cytometry data were analyzed using FlowJo software.
Murine ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Murine IFN-γ is a recombinant protein that represents the murine (mouse) form of interferon gamma (IFN-γ). IFN-γ is a cytokine that plays a critical role in the immune response. This product can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (7 mentions)
Murine il 4, by Thermo Fisher Scientific (6 mentions)
Pam3csk4, by InvivoGen (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tnf α, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Murine tnf α, by Thermo Fisher Scientific (2 mentions)
Human ifn γ, by Thermo Fisher Scientific (2 mentions)
Rprotein a sepharose bead, by GE Healthcare (2 mentions)
Octylglucopyranoside, by Merck Group (2 mentions)
Anti h 2kb antibody y3, by BioXCell (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Zymosan, by Merck Group (2 mentions)
Monensin, by Merck Group (2 mentions)
Bafa1, by Bio-Techne (2 mentions)
Pp242, by Bio-Techne (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (2 mentions)
53 protocols using murine ifn γ
1
Murine Peritoneal Macrophage Activation Assay
2
HLA-DR4 Transfection and Expression
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Cell lines were transfected using the Lipofectamine Transfection Reagent (Invitrogen) utilizing the protocol previously described.15 (link) B16F1, LLC/2 and Pan02 cells were transfected with constitutive HLA-DR4 using the pDC Vitro 2 chimeric HLA-DR401 plasmid. B16F1 cells were also transfected with the IFNγ-inducible HLA-DR4 using the pDC GAS chimeric HLA-DR401 plasmids where chimeric HLA-DR401 is under expression of the IFNγ-inducible promoter. Plasmid details have previously been described in full.15 (link)
Transfected cells were selected by growth in the presence of zeocin (300 μg/mL), or hygromycin B (300 μg/mL). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the HLA-DR PE conjugated antibody (clone L243, eBioscience). Cells transfected with the IFNγ-inducible plasmid were incubated overnight in the absence or presence of murine IFNγ (30 ng/mL, Gibco Life Technologies) before staining with the antibody.
Transfected cells were selected by growth in the presence of zeocin (300 μg/mL), or hygromycin B (300 μg/mL). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the HLA-DR PE conjugated antibody (clone L243, eBioscience). Cells transfected with the IFNγ-inducible plasmid were incubated overnight in the absence or presence of murine IFNγ (30 ng/mL, Gibco Life Technologies) before staining with the antibody.
3
Macrophage Infection Assay with Mycobacteria and Staphylococcus
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RAW cells or BMDMs were transfected with siRNAs for 2d (RAW) or 3d (BMDM) prior to infection. 200 U/ml murine IFN-γ (Gibco) was added 24h before infection as indicated. For Mtb and M. smegmatis, macrophages were infected with a single cell suspension at an MOI of 3 with at least three replicates per experiment as previously described [26 (link)]. 4 hpi macrophages were extensively washed, lysed with 0.1% Triton X-100 at indicated time points, and serial dilutions were plated on 7H11. CFU were counted 15–21 days later for Mtb and 2–3 days later for M. smegmatis. For S. aureus infection, bacteria were opsonized with human serum for 1h prior to infection. Macrophages were infected at an MOI of 1, washed extensively 30 min post-infection, and lysed in 0.1% Triton-X-100 at indicated time points. S. aureus were plated on Tryptic Soy Agar, and CFU were quantified the following day.
4
Genetically engineered melanoma and lung cancer cell lines
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B16F1 melanoma and LLC/2 lung carcinoma cells expressing HLA-DR4 under expression of the constitutive and IFNγ inducible promoters have been described previously.9 The B16F1 cell line previously knocked out for murine MHC class I and II by Zinc finger Technology (Sigma Aldrich) was transfected using Lipofectamine LTX with plus reagent (Invitrogen), with 4 µg of each plasmid, pCDNA3 HHDII in combination with either the pVITRO2 Human HLA-DP4 or pDCGAS Human HLA-DP4 plasmids, where DP4 is under expression of the constitutive or IFNγ inducible promoter, respectively. Transfected cells were selected by growth in the presence of G418 (500µg/ml) with either Hygromycin B (300µg/ml) or Zeocin (300µg/ml). The LLC/2 lung carcinoma cell line was also transfected with the pVITRO2 Human HLA-DP4 plasmid and selected on incubation with media supplemented with Hygromycin B (300µg/ml). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the anti-human beta 2 microglobulin FITC (clone TU19, BD Biosciences) and anti-human HLA-DR/DP/DQ (clone WR18, Abcam) PE antibodies. Cells transfected with the IFNγ inducible plasmid were incubated overnight in the absence or presence of murine IFNγ (50ng/ml, Gibco Life Technologies) prior to staining with the antibody.
5
Murine Macrophage Modulation by IFNγ and 3d
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RAW264.7 cells were seeded in tetraplicates in 96-well plates at a density of 105 cells/well and incubated under standard conditions for 24 h. The cells were treated with murine IFNγ (20 ng/mL) (Gibco, Grand Island, NY, USA) with or without (control) 3d (10–50 µM) for a further 24 h. At the end of the incubation period, the levels of TNF-α and NO in culture medium were measured using murine TNF alpha ELISA (Thermo Scientific, Frederick, MD, USA) and the Griess reagent system (Promega, Madison, WI, USA), respectively, according to manufacturers’ instructions.
6
Isolation and Culture of Bone Marrow-Derived Macrophages
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To culture bone marrow derived macrophages (BMDM), bone marrow was flushed from the femurs and tibias of mice with DM10 media (DMEM (Gibco) supplemented with 10% FBS, 1% sodium pyruvate, 1% l -glutamine, 1% penicillin/streptomycin). The bone marrow cells were cultured for 6 days in BM macrophage media containing 1% P/S (DMEM media supplemented with 10% Bovine Growth Serum, 1% sodium pyruvate, 1% l -glutamine, 50 μM 2-mercaptoethanol, 10% L-cell conditioned media). Cells were provided fresh media on Day 3 and on Day 6 BMDMs were harvested and plated without P/S for experiments to be conducted on Day 7. Peritoneal cells were isolated from naïve mice by injecting 10-mL of ice-cold PBS into the peritoneal cavity and were cultured in DM10 media. For flow cytometric analysis, peritoneal cells were cultured in non-tissue culture treated suspension plates (Gibco) to minimize cell adherence. Spleens were digested and disrupted into single cell suspension as previously described (Eshleman et al., 2017 (link)). To determine MHCII expression, cells were stimulated with 100 U/mL murine IFNγ (Life Technology, #PMC-4031) for 18–24 h. All cell cultures were maintained at 37 °C with 7.5% CO2.
7
Isolation and Characterization of Murine Peritoneal Cells
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Peritoneal cells were isolated from naïve mice by lavage using 10 mL of ice cold PBS from the peritoneal cavity. Non-tissue culture treated suspension plates were used to minimize cell adherence for experiments involving flow cytometric surface expression analysis. For experiments using western blot analysis, peritoneal cells were placed on tissue-culture treated plates for several hours to ensure adherence by the macrophages and non-adherent cells were removed by vigorous washes with PBS. Cells were cultured in DM10 media (DMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin). In experiments evaluating IFNGR1, cells were treated with 100 Units (U)/mL murine IFNβ (PBL, #12401–1) for 6–8 hrs. For experiments evaluating MHC II up regulation cells were treated ± 100 U/mL IFNβ for 6–8 hrs followed by treatment with 100 U/mL murine IFNγ (LifeTechnology, #PMC-4031) for 18–24 hrs. For MHC I expression, cells were treated with 100 U/mL IFNβ for 24 hrs.
8
Measuring Cytokine Levels in Cell Culture
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The cytokine concentration in cell culture supernatants was measured by sandwich ELISA. ELISA was performed in MaxiSorp plates (Nunc, Rochild, Denmark) with paired antibodies, according to the manufacturer’s instruction. The samples were analyzed in duplicates for murine TNF, murine IFN-γ, murine/rat IL-17 (all from eBioscience), murine IL-6 and rat IFN-γ (both from R&DSystems, Minneapolis, MN). For all the ELISA tests, the lower limit of detection was 30 pg/mL, while the upper limit of detection was 10 ng/mL. If the values over the upper limit were detected, the appropriate samples were adequately diluted for measurement. Absorbance was read using an automated microplate reader (LKB 5060-006). The results were calculated using standard curves, based on the absorbance of the known concentrations of recombinant cytokines.
9
IFN-γ Stimulation in Cell Lines
10
Protein Profiling of MSC Secretome
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Protein profiling of cell culture supernatant was undertaken using a Proteome Profiler (R&D Systems, USA). PαS MSC were cultured to passage 5, and some flasks were stimulated with 20 ng/mL of murine IFN-γ (PeproTech, UK) and 20 ng/mL murine TNF-α (PeproTech, UK). Protein profiling was carried out following the manufacturer’s instructions. Protein arrays contain internal positive and negative controls and undergo normalization across each blot allowing semi-quantitative representation of expression data. Blots were processed using x-ray imaging, and images were scanned and analyzed with ImageJ.
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