Purified CD4+ cells were stimulated with 4 μg/ml plate-bound anti-CD3 (eBioscience, Clone: 145-2C11) and 1 μg/ml soluble anti-CD28 (BD, Clone: 37.51;). For Th17 differentiation, cells were cultured in the presence of 5 ng/ml recombinant human TGFβ (R&D Systems), 20 ng/ml murine IL-6 (eBioscience), 10 μg/ml anti-IFNγ (eBioscience, Clone: XMG1.2), and 10 μg/ml anti-IL-4 (eBioscience, Clone 11B11). For Th1 differentiation, cells were cultured for up to 7 d with 10 ng/ml IL-12 (PeproTech) and 10 μg/ml anti-IL-4. For Treg induction purified mouse CD4+ T cells were stimulated with 1 ng/ml rhTGF-β for 72 h if not indicated differently. Cells were restimulated and intracellularly stained for flow cytometric analysis.
Murine il 6
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Murine IL-6 is a recombinant protein that represents the mouse homologue of the human cytokine interleukin-6 (IL-6). IL-6 is a pleiotropic cytokine involved in various biological processes, including the immune response, inflammation, and hematopoiesis.
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Lab products found in correlation
Murine il 3, by Thermo Fisher Scientific (9 mentions)
Murine scf, by Thermo Fisher Scientific (8 mentions)
Murine il 4, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Plasmocin, by InvivoGen (3 mentions)
Murine stem cell factor, by Thermo Fisher Scientific (3 mentions)
Anti ifn γ, by Thermo Fisher Scientific (2 mentions)
Anti il 4, by Thermo Fisher Scientific (2 mentions)
Sytox blue cell death dye exclusion, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa hts analyzer, by BD (2 mentions)
Human tgf β, by Thermo Fisher Scientific (2 mentions)
Tgf β, by R&D Systems (2 mentions)
Human flt3l, by Thermo Fisher Scientific (2 mentions)
Murine gm csf, by Thermo Fisher Scientific (2 mentions)
Recombinant human tgf β, by R&D Systems (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Soluble anti cd28, by BD (1 mentions)
24 protocols using murine il 6
1
T Cell Subset Differentiation Protocols
2
Sandwich ELISA for Cytokine Quantification
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Sandwich ELISA was used to measure the cytokine content in liver supernatants using MaxiSorp plates (Nunc, Rochild, Denmark). Anti-cytokine-paired antibodies were used for cytokine detection according to the manufacturer’s instructions. The antibodies were: anti-mouse TNF-α purified rabbit monoclonal (clone EPR16803-2), anti-mouse TNF-α biotinylated rabbit monoclonal (EPR16803-84, both from Abcam), anti-mouse IL-6 purified mouse monoclonal (clone MP5-20F3), and anti-mouse IL-6 biotinylated mouse monoclonal (MP5-32011, both from eBioscience). The absorbance was measured using multiplate reader Synergy H1 at 450 nm, with a correction at 690 nm. Standard curves, based on known concentrations of recombinant murine TNF (Abcam) and murine IL-6 (eBioscience) were used for determination of cytokine concentrations in samples and all ELISA test samples were analyzed in duplicates.
3
Murine Cytokine ELISA Protocol
4
Murine MLL-AF9 Leukemia Model
5
Modulation of Macrophage Polarization by NAMPT
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C269 (10 μg/mL), control IgG1 (10 μg/mL) and recombinant murine NAMPT (rNAMPT, 500 ng/mL) were produced and purified as previously described (Colombo et al., 2020 ). PECs were treated with LPS (100 ng/mL lipopolysaccharides from Escherichia coli O 111:B4, Sigma, Cat. No. L2630), murine IFNγ (Peprotech, 200 U/mL), murine IL-4 (Peprotech, 20 ng/mL), murine IL-6 (100 ng/mL), murine GM-CSF (50 ng/mL) and murine IL-1β (50 ng/mL). Stattic (Merck Life Science) was used at 3μM for 1 h.
For treatment with C269, a 6-multiwell plate was coated with the antibodies in a 100 mM of sodium bicarbonate solution O.N. After that, the plate was washed and incubated with medium containing eNAMPT at 37°C for 1 h. Then 0 ,4 μm Transwell Inserts seeded with PECs were added to the plate.
MDM were treated with human IFNγ (Peprotech, 200 U/mL) and/or murine NAMPT (Peprotech, 500 ng/mL).
For treatment with C269, a 6-multiwell plate was coated with the antibodies in a 100 mM of sodium bicarbonate solution O.N. After that, the plate was washed and incubated with medium containing eNAMPT at 37°C for 1 h. Then 0 ,4 μm Transwell Inserts seeded with PECs were added to the plate.
MDM were treated with human IFNγ (Peprotech, 200 U/mL) and/or murine NAMPT (Peprotech, 500 ng/mL).
6
Fetal Liver Cell Colony Assay
7
In Vitro Polarization of Murine CD4+ T Cell Subsets
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Naive CD4+CD44−CD62L+ T cells were resuspended at 1 × 106 cells/mL in RPMI 1640 supplemented with 10% HI-FCS, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomyocin and stimulated with plate-bound 1 μg/mL anti-CD3ε mAb and 1 μg/mL anti-CD28 (37.51, BioLegend) at 200 μL/well in a 96-well plate. Cells were polarized as indicated using the following culture conditions: Th0, 5 μg/mL anti-IL-4 mAb (11B11, BioLegend) and 5 μg/mL anti-IFN-γ mAb (XMG1.2, BioLegend); Th1, 10 ng/mL murine IL-12 (PeproTech) and 5 μg/mL anti-IL-4 mAb; Th2, 10 ng/mL murine IL-4 (PeproTech) and 5 μg/mL anti-IFN-γ mAb; Th17, 5 μg/mL anti-IL-4 mAb, 5 μg/mL anti-IFN-γ mAb, 10 ng/mL human IL-23 (PeproTech), 1 ng/mL human TGF-β (PeproTech), and 5 ng/mL murine IL-6 (PeproTech); Treg, 1 ng/mL human TGF-β, 5 μg/mL anti-IL-4 mAb, and 10 μg/mL anti-IFN-γ mAb. After 72 hr cells were analyzed by intracellular flow cytometry for CD4+ Th subset polarization.
8
Murine MLL-AF9 Leukemia Model
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Mouse MLL-AF9 leukemic cells with Dot1lfl/fl or wild-type genotypes were generated by transformation of mouse bone marrow Lin−Sca1+cKit+ (LSK) cells with retrovirus expressing MLL-AF9 fusion protein and transplanted into sublethally irradiated recipient mice as described previously8 (link). The leukemic blasts harvested from the diseased mice were cultured in vitro in IMDM plus 15% FBS supplemented with 20 ng/ml murine SCF (PeproTech), 10 ng/ml murine IL-3 (PeproTech) and 10 ng/ml murine IL-6 (PeproTech). Human leukemic cell lines Molm-13, MV4-11 and HL-60 were maintained in RPMI plus 10% FBS. All cell culture medium contained L-Glutamine (2mM; Gibco), penicillin (100 units/ml; Gibco), streptomycin (100 ug/ml; Gibco) and plasmocin (5 ug/ml; InvivoGen). Human cell lines including HL-60, MV4-11 and Molm-13 were tested in March - May, 2013 for authentication by short tandem repeat (STR) profiling performed by ATCC. Live cell counts were obtained by high-throughput flow cytometry with SYTOX-blue cell death dye exclusion (Life Technologies) using an LSRFortessa-HTS Analyzer (BD biosciences).
9
Coculture of HSCs with BMSCs
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2×105 lineage-negative cells (HSCs) were seeded over the BMSCs/M210B4 in IMDM supplemented with 20% Mesen-FBS (Stem Cell Technology), 25 ng/mL murine IL-6, 25 ng/mL murine stem cell factor and 10 ng/mL murine IL-3 (Peprotech, Rocky Hill, NJ), and the cocultures were maintained 7 days at 37°C under either normoxia (control-cocultures) or hypoxia (hypoxic-cocultures). Cocultures with CoCl2-BMSCs were incubated under normoxia (CoCl2-cocultures), unless stated otherwise.
After 7 days, the cocultures were harvested and viable cell counts were taken using the Trypan Blue dye exclusion method.
After 7 days, the cocultures were harvested and viable cell counts were taken using the Trypan Blue dye exclusion method.
10
Multiparameter Flow Cytometry Analysis
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Fluorescently conjugated protein or antibodies used for cell-surface staining and intracellular staining for cytokines and transcription factors are listed in Supplementary Table 2 . α-GalCer (KRN7000) was purchased from Enzo. CD1d-PBS57 (conjugated with either PE or allophycocyanin) was obtained from the tetramer facility of the US National Institutes of Health. Collagenase IV used for tissue digestion was purchased from Sigma. Taq Master Mix was purchased from Vazyme Biotech. A mouse IFN-γ ELISA kit and a mouse IL-4 ELISA kit were obtained from BioLegend. PMA and ionomycin were obtained from Merck. Stem cell factor, murine IL-3, and murine IL-6 were obtained from PeproTech. Met-RANTES was purchased from R&D Systems. DAPI was obtained from Cell Signaling Technology.
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