BMDMs were primed with recombinant mouse IFN-γ (10 ng/ml; BioLegend, San Diego, CA, USA) for 18 h before stimulation unless otherwise specified. M(IFNγ+LPS) and M(IFNγ+LPS+IC) were generated by adding Salmonella LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) or LPS in combination with IC. IC was prepared by adding a 10-fold molar excess of rabbit anti-OVA IgG to OVA (both from Sigma-Aldrich), and the complex was incubated for 30 min at room temperature (21 (link)). IC was used at a 1:100 volume ratio of IC to media for stimulation.
Recombinant mouse ifn γ
Manufactured by BioLegend
Sourced in United States
Recombinant mouse IFN-γ is a protein produced through recombinant DNA technology. It is a cytokine that plays a role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Cd43 microbead depletion, by Miltenyi Biotec (3 mentions)
Anti mouse igm f ab 2 fragment, by Jackson ImmunoResearch (3 mentions)
Anti mouse cd40, by Southern Biotech (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Lsrfortessa, by BD (2 mentions)
Rabbit anti ova igg, by Merck Group (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Nanoject 2, by Drummond (1 mentions)
Recombinant mouse tnf α, by R&D Systems (1 mentions)
Fludarabine, by Bio-Techne (1 mentions)
Mouse ip 10 elisa kit, by Thermo Fisher Scientific (1 mentions)
Human b cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Ifn β, by BioLegend (1 mentions)
Cd40l, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
Mouse il 6 elisa, by Thermo Fisher Scientific (1 mentions)
Raw264, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
25 protocols using recombinant mouse ifn γ
1
IFN-γ Primed Macrophage Polarization
2
Spinal Cord and Brain Cytokine Injections
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Stereotaxic injections were performed by Washington University's animal surgery core using a David Koft stereotaxic frame. Adult male and female Cxcr7GFP/+ animals were shaved and anesthetized with 2% isoflurane prior to surgery. The surgical site was cleaned with 75% ethanol, followed by 1% betadine. A 1.0 cm sagittal incision was made at the level of L2/L3 vertebrae for spinal cord injections or at the brain stem. A glass needle was inserted at the incision site using the Drummond Nanoject2. Stereotaxic injections were performed at a depth of 0.5 mm, just lateral to midline. A volume of 0.25 μl recombinant mouse TNFα (25 ng, R&D Systems), recombinant mouse IFNγ (25 ng, Biolegend), or vehicle (PBS) was injected. After microinjection, the dorsal muscle was sutured with 4–0 silk. The incision skin was pulled together using forceps and sutured using 4.0 nylon. The mice were kept on a heating pad until awake. Animals were sacrificed 72 hr postinjection.
3
IFN-γ-Induced CXCL10 Production
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2 x 104 cells were incubated with Fludarabine (Tocris Bioscience) at 37C for two hours in complete culture media. Then cells were stimulated with 1ng/ml of recombinant mouse IFN-γ (Biolegend) for 24 hours. Mouse CXCL10 protein concentration was measured from supernatants mouse IP-10 ELISA kit (Invitrogen) according to manufacturer’s protocol.
4
Murine and Human B Cell Activation
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For mice, splenic B cells were purified from WT, Ifnar−/−, Tbx21−/−, and Ifngr−/− mice by CD43 microbead depletion (Miltenyi Biotec). Purified B cells were cultured in RPMI at 37°C for 48 h at 106 cells/well in a 96-well plate with or without 5 ng/ml R848, 1 µg/ml anti–mouse IgM F(ab')2 fragment (Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml anti–mouse CD40 (SouthernBiotech), recombinant mouse IFN-γ or IFN-β (200 and 300 U/ml, respectively; BioLegend), and 500 nM ruxolitinib or 500 nM tofacitinib. For co-culture experiments, congenically marked CD45.1 WT and CD45.2 Ifngr−/− B cells were stimulated together in 96-well plates (106 total cells/well). B cell surface marker and transcription factor expression was evaluated by flow cytometry. Cell proliferation was evaluated by Cell Trace violet (Invitrogen) dilution.
Total human B cells were purified by the Human B Cell Isolation kit II (Miltenyi Biotec). Total B cells were plated at 5 × 104 in a 96-well plate for 24 or 72 h with 10 µg/ml anti-IgM (Jackson ImmunoResearch Laboratories, Inc.), 5 µg/ml CD40L (PeproTech), or 3.75 µg/ml R848 (InvivoGen) with or without 10 µg/ml IFN-γ (R&D Systems). For JAK inhibitor experiments, ruxolitinib or tofacitinib was added at 100 nm or 500 nm at the initiation of culture. Cells were analyzed at 24- and 72-h time points.
Total human B cells were purified by the Human B Cell Isolation kit II (Miltenyi Biotec). Total B cells were plated at 5 × 104 in a 96-well plate for 24 or 72 h with 10 µg/ml anti-IgM (Jackson ImmunoResearch Laboratories, Inc.), 5 µg/ml CD40L (PeproTech), or 3.75 µg/ml R848 (InvivoGen) with or without 10 µg/ml IFN-γ (R&D Systems). For JAK inhibitor experiments, ruxolitinib or tofacitinib was added at 100 nm or 500 nm at the initiation of culture. Cells were analyzed at 24- and 72-h time points.
5
Murine Splenic B Cell Activation
6
Splenic B Cell Activation Assay
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Splenic B cells were purified from WT, Ifngr−/−, Tbx21−/−, and Stat1−/− mice by CD43-microbead depletion (Miltenyi Biotec). Purified B cells were cultured in RPMI-1640 medium (supplemented with 10% FCS, 1% penicillin-streptomycin, sodium pyruvate, Hepes, glutaMAX, and 0.1% β-ME) at 37°C for 48 h. B cells were seeded at a density of 106 cells/well in a 96-well plate with or without R848 (5 ng/ml); anti–mouse IgM F(ab′)2 fragment (1 µg/ml, Jackson ImmunoResearch, Inc.); anti–mouse CD40 (1 µg/ml, SouthernBiotech); recombinant mouse IFN-γ (200 U/ml, BioLegend); and ruxolitinib (500 nM) or tofacitinib (500 nM). B cell surface markers were evaluated by flow cytometry. Cell proliferation was evaluated by Cell Trace Violet (Thermo Fisher Scientific) dilution. IL-6 in the supernatants and serum was measured by mouse IL-6 ELISA (eBioscience).
7
Macrophage activation by amelogenin
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The THP-1 human monocytic cell line and the RAW264.7 murine macrophage cell line were purchased from RIKEN BioResouce Center (Ibaraki, Japan). THP-1 cells were maintained in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin, and streptomycin at 37°C in a 5% CO2 incubator. Cells were sub-cultured every 48–72 h, inoculum being 5 × 105/mL, and cell viability (>95%) was confirmed by trypan blue exclusion. After the THP-1 cells were stimulated with 50 nM phorbol-myristate-acetate (PMA) (Sigma Aldrich, St. Louis, MO, USA) for 24 h, they were washed with phosphate-buffered saline (PBS, pH 7.4) and replaced with PMA-free RPMI-1640 medium with FBS for another 24 h. These differentiated macrophages were pretreated with or without 10 μg/mL recombinant murine M180 amelogenin (rM180) for 24 h, and then stimulated with 2.5 ng/mL recombinant human IFNγ (PeproTech, Rocky Hill, NJ, USA) for 24, 36, and 48 h. The murine macrophage cell line RAW264.7 (American Type Culture Collection (ATCC), Manassas, VA, USA) was maintained in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) containing 10% heat-inactivated FBS, penicillin, and streptomycin. RAW264.7 cells were stimulated in same manner as described for THP-1 cells using 2.5 ng/mL recombinant mouse IFNγ (BioLegend, San Diego, CA, USA) and 10 μg/mL rM180.
8
Analyzing PD-L1 expression in IFN-γ-treated mice
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Recombinant mouse IFN-γ (5×104 IU; BioLegend) was injected intravenous (i.v.) daily for 4 days. IFN-γ-treated animals were then subjected to PET/CT imaging and ex vivo biodistribution or lung tissue analysis by FACS. For FACS analysis, the lungs of treated mice were extracted, perfused via the right ventricle and rinsed with PBS to remove blood cells. Single-cell suspensions were generated with a Miltenyi lung dissociation kit according to the manufacturer's instructions. Cells were further stained with fluorochrome-conjugated mAbs, and PD-L1 expression was analyzed on a BD FACSVerse flow cytometer.
9
Membrane Labeling and Cell Sorting
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For membrane labeling, TILs and cancer cells were stained with fluorochrome-coupled mAbs incubated for 20 minutes at 4°C, protected from light, and then washed with 1× PBS. Intracellular staining was performed after permeabilization with the FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, catalog 00-5523-00) and intracellularly labeled with anti-Foxp3 mAb (eBiosciences, clone PCH101) and anti-Ki67 (BD Biosciences, catalog 556027), following the manufacturer’s protocol. Cell samples were acquired on a BD LSRFortessa X-20 flow cytometer (BD Biosciences) with single-stained Ab-capturing beads used for compensation (CompBeads, BD Biosciences, catalog 552843). For cell sorting, Zombie Aqua– cells and anti-HLA-I+ and HLA-I– cells after BCG coincubation for 24 hours were sorted on a BD FACSAria III Fusion (BD Biosciences). Data were analyzed with Kaluza Analysis software, version 2.1 (Beckman Coulter). MB49 and UPPL1541 cell lines were cultured in vitro into media with or without recombinant mouse IFN-γ (BioLegend, catalog 575306) for 24 hours and subsequently stained for HLA-I with FITC anti-mouse H-2Kb Ab (BioLegend, catalog 1165005) and run by flow cytometry.
10
Acalabrutinib regulates B cell and macrophage activation
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1 x 105 cells of MZ B cells and FO B cells from spleen, splenic Mac, and PEC Mac were stimulated with 10 μg/ml LPS (O55:B5, Sigma-Aldrich) for 6 h or 24 h in 96 well plate at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib or, 1 μM BAY 11-7082 (Selleck Chemicals). CD43- B cells were labeled with 20 μM Cell Trace Violet (Invitrogen) for 5 min at R.T. The cells were stimulated with anti-IgM F(ab’)2 (Jackson), anti-CD40 (HM40-3, BD Biosciences), recombinant mouse IL-2 (R&D), IL-4 (R&D), IL-5 (R&D) for 72 h or 96 h in 48 well plates at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib. Acalabrutinib was dissolved in DMSO and diluted with culture medium (Supplementary Methods
1 x 105 cells of BMDM were stimulated with 1 μg/ml LPS (O55:B5, Sigma-Aldrich) and 20 ng/ml recombinant mouse IFN-γ (BioLegend) for 6 h or 24 h in 96 well plates at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib.
1 x 105 cells of BMDM were stimulated with 1 μg/ml LPS (O55:B5, Sigma-Aldrich) and 20 ng/ml recombinant mouse IFN-γ (BioLegend) for 6 h or 24 h in 96 well plates at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib.
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