2Antibodies against GAPDH sc-322 (Santa Cruz Biotechnology, Santa Cruz, CA), pSTAT1 Tyr701 #9167, pS6 #15967, pAkt Ser473 #4060, Akt1 #2967, pGSK3β #5558, pH2AX (γ-H2AX) mAb #9718, pp53 #9286, CHK1 # 2348 and pBRCA1 #9009 were obtained from Cell Signaling Technology (Danvers, MA). F4/80 Monoclonal Antibody (BM8) coupled to Biotin (eBioscience, Waltham, MA) was used according to manufacturer's instructions. p-Histone H3, ab32107 was obtained from Abcam (Cambridge, MA). Alexa conjugated antibodies were obtained from Thermo Fisher Scientific (Waltham, MA). AffiniPure goat and rabbit anti-horseradish peroxidase (HRP) were obtained from Jackson Immunoresearch (San Diego, CA) and 4′,6-Diamidino-2-phenylindole (DAPI) SC-3598 from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant mouse IFNγ (PeproTech, Rocky Hill, NJ) was dissolved in 0.002% mouse serum albumin (MSA; Sigma-Aldrich, St. Louis, MO) and used at 2.5 μg/kg of weight. LiCl (Sigma-Aldrich, St Louis, MO) was used at 4 mg/mice and 4 μM in vitro. Rapamycin used as previously reported by us [14] (link) Doxorubicin was obtained from Pfizer and used at 3.45 μM. AZD8055 and XAV939 were used as previously reported by us [16] (link), [17] . Dextran sulfate sodium YD318041799 (Carbosynth, San Diego, CA) was dissolved at 3% in drinking water.
Recombinant mouse ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant mouse IFN-γ is a laboratory reagent that is produced through recombinant DNA technology. It is a cytokine that plays a role in immune system modulation.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Recombinant mouse tnfα, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Z vad fmk, by Merck Group (3 mentions)
Recombinant human g csf, by Amgen (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Thrombin, by Enzyme Research (3 mentions)
Human fibrinogen, by Enzyme Research (3 mentions)
Pstat1 tyr701 9167, by Cell Signaling Technology (2 mentions)
P akt ser473 4060, by Cell Signaling Technology (2 mentions)
Akt1 2967, by Cell Signaling Technology (2 mentions)
Alexa conjugated antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dapi sc 3598, by Santa Cruz Biotechnology (2 mentions)
Tgtp sc 11079, by Santa Cruz Biotechnology (2 mentions)
Lrg 47 sc 11075, by Santa Cruz Biotechnology (2 mentions)
Gtpi sc 11088, by Santa Cruz Biotechnology (2 mentions)
Recombinant mouse tnf, by BioLegend (2 mentions)
Sb202190, by Merck Group (2 mentions)
85 protocols using recombinant mouse ifn γ
1
Comprehensive Immunoblotting Protocol
2
Mouse IFNγ for Pneumocystis Infection
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Recombinant mouse IFNγ (2 μg per dose, Peprotech) was administered intra-tracheally on days−3 and−1 prior to infection with 5e5 freshly isolated mouse Pneumocystis organisms, and then every 3 days thereafter for the duration of the study.
3
Antibody Procurement for Parasite Research
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Goat polyclonal antibody against Irgb6 (TGTP; sc-11079) was purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti-GBP2 and mouse monoclonal anti-p62 (H00008878-M01J) antibodies were obtained from Proteintech and Abnova, respectively. An anti-ubiquitin rabbit monoclonal antibody (Apu2; Merck) was obtained from Nippon Biotest Laboratories. A mouse monoclonal anti-Irga6 (10D7) antibody was provided by J.C. Howard (Instituto Gulbenkian de Ciencia). Mouse monoclonal anti-GRA2 antibody and rabbit polyclonal anti-GAP45 antibody were provided by D. Soldati-Favre (University of Geneva). A rabbit polyclonal anti-GRA7 antibody was provided by John C. Boothroyd (Stanford University). Recombinant mouse IFN-γ was purchased from PeproTech.
4
Eosinophil-Mediated T Cell Modulation
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Flow-cytometry-purified BM-Eos (B6J) or magnetically enriched splenic eosinophils (Il5-tg) or A-Eos and B-Eos sorted from the GI tract (Il5-tg) were isolated from 6–12-week-old female and male mice. BM-Eos or spleen-derived eosinophils were conditioned overnight with colon CM (1:10) or treated with recombinant mouse IFNγ (10 ng ml−1, PeproTech) and/or IL-33 (20 ng ml−1, PeproTech), as indicated. Naive CD4+ T cells were isolated from the lymph nodes of 6–12-week-old female and male mice (B6J), enriched with the MojoSort Mouse CD4 Naïve T Cell Isolation Kit (480040 BioLegend) and purified by flow cytometry. T cells were labelled with the CellTrace CFSE Cell Proliferation Kit (C34554 Thermo Fisher Scientific) following the manufacturer’s instructions. T cells were then activated by CD3/CD28 T-activator Dynabeads (11131D Gibco) and co-cultured with eosinophils at a 1:1 ratio (2 × 105 total) for 4 days at 37 °C in complete RPMI medium supplemented with 10 ng ml−1 recombinant mouse IL-5 (PeproTech) and 20 ng ml−1 IL-2 (402-ML R&D). CFSE dilution was assessed by flow cytometry.
5
In Vitro Activation and Cytotoxicity Assay
6
Cytokine and Chemokine Quantification in Murine Immune Cells
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To detect IFN-γ, IL-2, IL-4, or IL-17A protein levels, 2 × 105 mononuclear cells or 105 CD4+ T cells from the indicated organs of pAAV- or pAAV-HBV1.2–injected mice were separated and cultured in 96-well U-bottomed plates in the presence of 2 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 for 96 h. In some indicated cases, CD4+ T cells (105) were stimulated with 2 µg/ml HBcAg (ID LABS) or 5 µg/ml HBsAg (Hytest) in the presence of autologous irradiated splenocytes for 96 h. Supernatants were then collected, and cytokine levels were measured using their corresponding CBA kit (BD) according to the manufacturer’s instructions. To detect CXCL9, a total of 105 hepatocytes, LNPCs, F4/80+ Kupffer cells, or F4/80− LNPCs were cultured for 24 h; in some cases, the cells were cultured in the presence of 50 ng/ml recombinant mouse IFN-γ (PeproTech). Supernatants were also collected to measure CXCL9 protein levels using a CXCL9 CBA kit (BD). To measure the CXCL9 levels in liver, tissues were removed, weighed, and homogenized for CBA assay, as previously described (Hou et al., 2009 (link)).
7
Cytokine-Conditioning of Neural Stem Cells
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NSCs were plated in CGM with or without either Th1-like (200 U/ml recombinant mouse TNF-α, Pepro Tech Inc.; 500 U/ml recombinant mouse IFN-γ, Pepro Tech Inc; 100 U/ml recombinant mouse IL-1β, Pepro Tech Inc) or Th2-like (10 ng/ml recombinant murine IL-4, R&D; 10 ng/ml recombinant mouse IL-5, R&D; 10 ng/ml recombinant mouse IL-13, R&D) cytokine cocktails for 16 h in vitro [33 (link)]. At the end of the conditioning, NSCs were washed three times with phosphate-buffered saline (PBS) to remove cytokine contamination before cell harvesting. Finally, NSCs and NSC-conditioned media were processed according to the analysis to be performed.
8
Cerebellum Slice Culture Protocols
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Cerebellum slice cultures were performed according to a previous publication.
23 (link) Irradiated or nonirradiated cerebella were embedded in 3% tissue culture grade agarose, and 300 μm sagittal slices were cut using a VT100S vibrating microtome (Leica). Slices were then transferred to a 0.4 μm Nuclepore membrane (Millicell) at the interface between air and culture medium (NB‐B27 medium) in a 6‐well culture plate and incubated at 37°C in 5% CO2. In certain experiments, cerebellum slices derived from nonirradiated mice were treated with 200 U/mL recombinant mouse IFN‐γ (Peprotech) for 24 h during culture, and then tissue lysates were prepared for Western blotting and qPCR. Cerebellum slices derived from irradiated mice 12 h post irradiation were treated with an IFN‐γ neutralizing antibody (clone R4‐6A2, BioXcell) or isotype control rat IgG1 for 24 h at 10 μg/mL, and then tissue lysates and frozen sections were prepared for Western blotting and immunohistochemistry, respectively.
23 (link) Irradiated or nonirradiated cerebella were embedded in 3% tissue culture grade agarose, and 300 μm sagittal slices were cut using a VT100S vibrating microtome (Leica). Slices were then transferred to a 0.4 μm Nuclepore membrane (Millicell) at the interface between air and culture medium (NB‐B27 medium) in a 6‐well culture plate and incubated at 37°C in 5% CO2. In certain experiments, cerebellum slices derived from nonirradiated mice were treated with 200 U/mL recombinant mouse IFN‐γ (Peprotech) for 24 h during culture, and then tissue lysates were prepared for Western blotting and qPCR. Cerebellum slices derived from irradiated mice 12 h post irradiation were treated with an IFN‐γ neutralizing antibody (clone R4‐6A2, BioXcell) or isotype control rat IgG1 for 24 h at 10 μg/mL, and then tissue lysates and frozen sections were prepared for Western blotting and immunohistochemistry, respectively.
9
Modulating Immune Responses with IFNγ and Gal-9
10
Generation and Genotyping of WNK4 Mice
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The generation of the WNK4D561A/+ mice and their genotyping strategies were described previously48 (link). Studies were performed on each strain using littermates. C57BL/6 mice were purchased from CLEA Japan. The mice were fed a normal diet [0. 4% NaCl (w/w)] or a high salt diet [8.0% NaCl (w/w)] (Oriental Yeast, Japan), and plain drinking water for seven days. In some experiments, to trigger the production of IFNγ inducible chemokines, intraperitoneal injection of recombinant mouse IFNγ (0.3 g/kg, Peprotech) was performed 3 h before organ collection. This experiment was approved by the Animal Care and Use Committee of the Tokyo Medical and Dental University and was performed in accordance with the guidelines for animal experiments of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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