For induction of GC B-cells in vitro, B-cells were isolated from mouse spleens using FACS or a mouse B-cell isolation kit (Miltenyi) and co-cultured with CD40LB fibroblast feeder lines in RPMI 1640 medium supplemented with 10% FBS, 5.5 × 10—5 M 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 1 mM essential amino acids, 100 U/mL of penicillin, and 100 µg/mL streptomycin. Mouse IL-4 (1 ng/mL; BioLegend) was added to primary co-culture for 4 days. Induced GC B-cells were then replated onto a fresh CD40LB feeder line and cultured with mouse IL-21 (10 ng/mL; BioLegend) or Mouse IL-4 for another 3 to 4 days.
Mouse il 4
Manufactured by BioLegend
Sourced in United States
Mouse IL-4 is a recombinant cytokine that corresponds to the mature form of mouse interleukin-4. It is a key regulator of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Mouse il 6, by BioLegend (3 mentions)
Mouse b cell isolation kit, by Miltenyi Biotec (2 mentions)
Mouse il 21, by BioLegend (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Easysep mouse cd4 positive selection kit 2, by STEMCELL (1 mentions)
Soluble anti cd3, by BioXCell (1 mentions)
Anti ifn γ, by BioXCell (1 mentions)
Anti il 12, by BioXCell (1 mentions)
Anti il 4, by BioXCell (1 mentions)
Anti cd28, by BioXCell (1 mentions)
Mouse il 12, by Thermo Fisher Scientific (1 mentions)
Human tgf β, by Thermo Fisher Scientific (1 mentions)
Click s medium, by Irvine Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Mouse csf 1, by BioLegend (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
Protein g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Methacholine mch, by Merck Group (1 mentions)
11 protocols using mouse il 4
1
In vitro Induction of Germinal Center B-cells
2
In vitro Induction of Germinal Center B-cells
3
Isolation and Polarization of Mouse CD4+ T Cells
4
Isolation and Differentiation of Murine BM-derived Macrophages
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Mouse BM-derived macrophages (BMDMs) were isolated21 (link). Freshly isolated femur and tibia from WT C57BL/6 mice were flushed with RPMI-1640 medium (Life Technologies). Cells were collected and passed through a 40 μm strainer. Red cells were depleted with ACK lysis buffer (Thermo Fisher). BM cells were cultured in RPMI-1640 medium supplemented with 5% FBS (Life Technologies). Cells were incubated with 10 ng/ml mouse CSF-1 (Biolegend, 576404) for 3 days, to induce macrophage differentiation, followed by treatment with 10 ng/ml mouse CSF-1 in the presence or absence of 100 ng/ml mouse IL-4 (Biolegend, 574306) or IL-6 (Biolegend, 575708) for 4 days.
5
Characterization of Allergic Response Markers
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SHL lyophilized powder for injection was provided by Hayao Pharmaceutical Co., Ltd. (Harbin, Heilongjiang, China). Mouse IL-4, IL-5, eotaxin, and total IgE (tIgE) ELISA kits were from Biolegend Co. (San Diego, CA, USA). Mouse IL-13 ELISA kit was obtained from Excell Technology Co. (Shanghai, China). Mouse mast cell protease-1 (mMCP-1) ELISA kit was purchased from Thermo Fisher Scientific (CA, USA). Aluminium hydroxide gel was from Chemtrade LLC. (Berkeley, CA, USA). Methacholine (Mch) was obtained from Sigma-Aldrich (St. Louis, MO, USA). O-phenylenediamine-dihydrochloride (OPD) was from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Protein G PLUS-Agarose was obtained from Santa Cruz Biotechnology (CA, USA). The HRP-labeled rat anti-mouse IgE antibody was from Abcam Co. (Cambridge, UK). All other reagents were of analytical grade.
6
Generating Bone Marrow-Derived DCs
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Bone marrow cells were flushed from the tibia of either IRAK-M−/− NOD or WT NOD mice. Cells were resuspended by repeated pipetting in culture medium, and erythrocytes were lysed before the culture. Bone marrow cells were then counted and seeded in 60-mm culture dishes at a density of 1.5 × 106/mL in DC differentiation medium (RPMI-1640 complete medium) with 5% FCS, 20 ng/mL mouse IL-4 (Biolegend), and 20 ng/mL mouse GM-CSF (Biolegend) for 7 days at 37°C. Culture medium was replenished every other day. Cells were harvested on day 7, were analyzed by flow cytometry after staining with CD11b and CD11c, and >90% of cells were CD11c+, which were considered to be bone marrow–derived DCs (BMDCs).
7
Isolation and Culture of Mouse T Cells
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Mouse IL-2 (catalog 575402), mouse IL-4 (catalog 547302), mouse IL-33 (catalog 580502), anti-mouse CD3ε (145-2C11), anti-mouse CD28 (37.51), human TGF-β1 (catalog 781802), anti-mouse IL-4 mAb (clone 11B11), anti-mouse IFN-γ mAb (XMG1.2), and Apotracker Green were purchased from BioLegend. Antibodies for TRAF4 (D1N3A), MyD88 (D80F5), p-mTOR (S2448, D9E), mTOR (7C10), p-AKT (S473, D9E), p-AKT (T308, D25E6), AKT (11E7), p-JNK (T183/Y185, 81E11), SAPK/JNK (no. 9252), p-p38 (T180/Y182, D3F9), p38 (D13E1), p-ERK1/2 (T202/Y204, D13.14.4E), Erk1/2 (137F5), p-IκBα (S32, 14D4), IκBα (L35A5), TRAF6 (D21G3), and β-Actin (8H10D10) were obtained from Cell Signaling Technology. Mouse T1/ST2 antibody (DJ8) and rabbit polyclonal against ST2 (ab228543) were obtained from MD Bioproducts and Abcam, respectively. AKT inhibitor VIII (CAS 612847-09-3) and LY3214996 (CAS 1951483-29-6) were purchased from Cayman Chemical. Primary mouse CD4+ T cells were cultured in complete TexMACS Medium (Miltenyi Biotec, 130-097-196) supplemented with 10% FBS, 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. CD8+ cells and ILCs were isolated from spleen using MojoSort Mouse CD8 T Cell Isolation Kit and Biotin anti-mouse Lineage Panel from BioLegend.
8
Culturing HEK293T and Primary B Cells
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Cells were cultured in a sterile incubator that was maintained at 37°C and 5% CO2. HEK293T cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. Primary splenic B cells were cultured in complete RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 50 µM β-mercaptoethanol, and the indicated stimulants. All stimulants, including anti-IgM (goat F(ab′)2 anti-mouse IgM; Southern Biotech), lipopolysaccharide (055:B5; Sigma-Aldrich), CpG (ODN 1826; Invivogen), mouse IL-4 (BioLegend), mouse CD40 ligand (eBioscience), phorbol-12-myristate-13-acetate (PMA; EMD Millipore), and ionomycin (EMD Millipore), were prepared as per manufacturer’s instructions and used at the concentration indicated in the text. For proliferation assays, the WST-1 cell proliferation reagent (Roche) was used as per manufacturer’s protocol.
9
Activation and Differentiation of Immune Cells
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The following conditions were used for B1 cell and CD4+ T cell stimulation. Enriched B1 cells were activated using 2.5 mg/mL LPS (ultrapure, Invivogen) in the presence of mouse IL-4, mouse IL-5 and mouse IL-6 (each 100 ng/mL; Biolegend) for 24 h at 37 C. Naı ¨ve CD4+ T cells were activated with ionomycin (1 mM; Life technologies) and phorbol 12-myristate 13-acetate (PMA), (10 ng/mL; Sigma Aldrich) or by incubation with surface-bound antibodies against CD3ε and CD28 (Biolegend). For surface coating of 96-well plates, 100 mL antibody mix containing 1 mg of each antibody in PBS was used per well and incubated for 2-3 h at 37 C followed by a washing step with 200 mL PBS.
Naı ¨ve CD4+ T cells were differentiated into FoxP3-expressing T cells by cultivation in anti-CD3ε/CD28-coated 96-well plates in the presence of human IL-2 (20 ng/mL; Biolegend), mouse IL-7 (100 ng/mL; Biolegend), mouse IL-15 (100 ng/mL; Biolegend), human TGF-beta1 (5 ng/mL; Biolegend) and retinoic acid (10 nM; Sigma-Aldrich) at 37 C for at least 48 h. FoxP3-expression was confirmed by flow cytometry after intracellular staining using the BD Cytofix/Cytoperm Kit and anti-FoxP3 antibody (clone MF-14; Biolegend).
Naı ¨ve CD4+ T cells were differentiated into FoxP3-expressing T cells by cultivation in anti-CD3ε/CD28-coated 96-well plates in the presence of human IL-2 (20 ng/mL; Biolegend), mouse IL-7 (100 ng/mL; Biolegend), mouse IL-15 (100 ng/mL; Biolegend), human TGF-beta1 (5 ng/mL; Biolegend) and retinoic acid (10 nM; Sigma-Aldrich) at 37 C for at least 48 h. FoxP3-expression was confirmed by flow cytometry after intracellular staining using the BD Cytofix/Cytoperm Kit and anti-FoxP3 antibody (clone MF-14; Biolegend).
10
Lung Cytokine Profiling in Mice
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The collected right lungs were digested in 1 mL of RIPA buffer (Merck-Sigma)
supplemented with EDTA-free protease inhibitor (complete Mini, Roche)
and homogenized (10 min at 50 Hz) using 5 mm stainless steel beads
in a Tissue Lyser (Qiagen). Lysates were centrifuged for 5 min at
2600g (Hettich GmbH), and supernatants were stored
at −80 °C until analysis. The total protein contents were
measured using a BCA assay (Pierce, Thermo Fisher Scientific) and
IL-1α, IL-1b, IL-6, TNF-α, MCP-1, GM-CSF, IL-17A, IL-23,
IL-12p70, IFN-γ, IFN-β, IL-27, and IL-10 concentrations
were assessed using a multiplex Mouse Inflammation Panel (13-plex,
v-plate, Biolegend) according to the manufacturer’s instructions
and using a BD FACS Verse flow cytometer (BD Biosciences). The concentration
of each sample was determined using standard curves. IL-4 concentration
in lysed lungs was determined using a single ELISA kit (mouse IL-4,
Biolegend). Cytokine concentrations were expressed in pg/mg of protein
by normalizing the obtained concentration to the total protein measured
using the BCA assay.
supplemented with EDTA-free protease inhibitor (complete Mini, Roche)
and homogenized (10 min at 50 Hz) using 5 mm stainless steel beads
in a Tissue Lyser (Qiagen). Lysates were centrifuged for 5 min at
2600g (Hettich GmbH), and supernatants were stored
at −80 °C until analysis. The total protein contents were
measured using a BCA assay (Pierce, Thermo Fisher Scientific) and
IL-1α, IL-1b, IL-6, TNF-α, MCP-1, GM-CSF, IL-17A, IL-23,
IL-12p70, IFN-γ, IFN-β, IL-27, and IL-10 concentrations
were assessed using a multiplex Mouse Inflammation Panel (13-plex,
v-plate, Biolegend) according to the manufacturer’s instructions
and using a BD FACS Verse flow cytometer (BD Biosciences). The concentration
of each sample was determined using standard curves. IL-4 concentration
in lysed lungs was determined using a single ELISA kit (mouse IL-4,
Biolegend). Cytokine concentrations were expressed in pg/mg of protein
by normalizing the obtained concentration to the total protein measured
using the BCA assay.
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