At sacrifice, BALF cells, lung, and CNS tissue were collected and prepared as previously described (Kim et al., 2019 (link); Nguyen et al., 2019 (link)). Briefly, the cells were stained with anti-CD4 (RM4–5, BioLegend), anti-Ly6G (1A8, BD Biosciences), and anti-Siglec-F (1RNM44N, eBioscience) mAbs. For intracellular staining, cells were stained with anti-IL-4 (11B11, eBioscience), anti-IL-13 (eBio13A, eBioscience), anti-IL-17 (TC11–18H10, BD Bioscience), anti-IFN-γ (XMG1.2, BD Bioscience), anti-IL-5 (TRFK5, BD Biosciences), anti-IL-10 (JES5–16E3, BD Biosciences) and anti-IL-2 (JES6–5H4, BD Biosciences) mAbs. Cells were acquired using a FACSLSRII or FACSFortessa X-20 (BD Biosciences) and analyzed using a FlowJo software (Tree Star Inc.). For intracellular staining, cells were harvested separately and stimulated ex vivo with PMA (10ng/mL, Millipore-Sigma) and ionomycin (1 μM, Millipore-Sigma) for 4 h in the presence of 2 μM monensin (Calbiochem) during the last 2 h of stimulation. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence-conjugated mAbs.
Anti il 4 11b11
Manufactured by Thermo Fisher Scientific
Anti-IL-4 (11B11) is a monoclonal antibody that specifically binds to interleukin-4 (IL-4), a cytokine involved in immune regulation. This antibody can be used in various research applications to study the role of IL-4 in biological processes.
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Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Il 12, by Thermo Fisher Scientific (3 mentions)
Anti il 13 ebio13a, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Anti cd16 cd32 fc block 93, by Thermo Fisher Scientific (2 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti il 17 tc11 18h10, by BD (1 mentions)
Anti il 5 trfk5, by BD (1 mentions)
Anti il 10 jes5 16e3, by BD (1 mentions)
Anti il 2 jes6 5h4, by BD (1 mentions)
Facs fortessa x20, by BD (1 mentions)
Anti ly6g 1a8, by BD (1 mentions)
Anti ifn γ xmg1.2, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Facs lsr 2, by BD (1 mentions)
Anti cd4 rm4 5, by BioLegend (1 mentions)
Anti gapdh 14c10 antibodies, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 mouse anti stat5 py694, by BD (1 mentions)
12 protocols using anti il 4 11b11
1
Multiomics analysis of immune responses
2
Comprehensive Immune Cell Profiling
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FITC- or Pacific Blue-conjugated anti-mouse CD4 (GK1.5), Brilliant Violet 510™-conjugated anti-mouse CD4 (MR4-5), APC-conjugated anti-mouse CD45.1 (A20), FITC-conjugated anti-mouse TNF-α (MP6-XT22), APC-conjugated anti-mouse CTLA4 (UC10-4B9), and APC-conjugated anti-mouse GITR (DTA-1) antibodies were purchased from Biolegend. PE-conjugated, APC-conjugated, or Pacific Blue-conjugated anti-mouse Foxp3 antibodies (FJK-16S); PerCP-Cy5.5-conjugated anti-mouse CD45.1 (A20), PE-conjugated anti-mouse PD-1(J43), FITC-conjugated anti-mouse Ki67 (SolA15), APC-conjugated anti-mouse IFN-γ (XMG1.2), anti-IFN-γ (R4-6A2), and anti-IL-4 (11B11) antibodies were purchased from eBioscience. Alexa Fluor 647 Mouse anti-STAT5 (pY694) and PE-conjugated anti-STAT3 (pY705) antibodies were purchased from BD bioscience. Anti-Phospho SMAD3 (phosphor S423+425′ EP823Y) and anti-SMAD3 (AF9F7) antibodies were purchased from Abcam. Anti-GAPDH (14C10) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
T cell activation and Th1 polarization
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For early activation markers, cells were incubated for 24h on plate-bound anti-CD3e (10 mg/ml) and anlalyzed by flow citometry. For phosphorylated molecules downstream of TCR vs isotype-matched control antibody, sorted lymph node cells were stimulated for 5 min with soluble anti-CD3ε (10 μg/ml) then fixed 30min at 4°C and permeabilized with the Foxp3/Transcription Factor Staining Buffer set (eBioscience) in the presence of anti CD16/CD32 Fc Block (93) (eBioscience) for 30min at room temperature, and finally incubated for 1h at room temperature with identified above anti-p-ERK Abs in permeabilization buffer. For Th1 polarization CD4+ αβ T cells were isolated by flow cytometry and incubated for 4 days on plate-bound anti-CD3ε and anti-CD28 (5ng/ml each). When indicated (Th1 cocktail), the following cytokines and neutralizing antibody were added to the culture milieu: IL-2 (10 ng/ml; Preprotech), IL-12 (50 ng/ml; Preprotech) and anti-IL-4 (11B11) (10 μg/mL; eBiosciences).
4
Naive CD4+ T Cell Differentiation
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Naive CD4+ T (CD44lowCD62LhighCD25−) cells were purified using a CD4+ naive T cell negative isolation kit according to the manufacturer's protocol (STEMCELL Technologies). The in vitro differentiation experiments were performed as previously described. Naive CD4+ T cells were stimulated with immobilized anti-CD3 (5 μg/ml; 145-2C11; eBioscience) and anti-CD28 (5 μg/ml; 37.51; eBioscience) for 2 days. Then, the cells were washed and transferred to a new plate and further expanded in medium with hIL-2 (50 U/ml, R&D Systems) for 2 days. For Tfh-like cell differentiation, naive CD4+ T cells were activated with anti-CD3 and anti-CD28 as above and treated with 20 ng/ml IL-6 (R&D Systems), 20 ng/ml IL-21 (R&D Systems), 10 μg/ml anti-IL-4 (11B11, eBioscience), 10 μg/ml anti-IFN-γ (XMG1.2, eBioscience), and 20 μg/ml anti-TGF-β (1D11, R&D Systems) for 4 days.
5
T cell activation and Th1 polarization
6
Polarization of Naive CD4+ T Cells
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Naïve CD4+ T cells were cultured (3x105 cells/well) in 96-well plates coated with anti-CD3 mAb (5 μg/ml) and soluble anti-CD28 mAb (1 μg/ml) and kept in presence of IL-2 (100 U/ml). For TH0 conditions anti-IL-4 (10 μg/ml) and anti-IFN-γ (10 μg/ml) mAb were added. Cultures were supplemented with IL-12 (10 ng/ml) and anti-IL-4 mAb (10 μg/ml) for TH1 polarization; with IL-4 (20 ng/ml), anti-IFN-γ (20 μg/ml) and anti-IL-12 (10 μg/ml) for TH2 polarization; and with TGF-β1 (5 ng/ml), anti-IFN-γ, anti-IL-4 mAb and anti-IL-12 (all 10 μg/ml) for induced Treg polarization. Sorted Treg cells were activated for 48h with anti-CD3 mAb (5 μg/ml) and soluble anti-CD28 mAb (1 μg/ml) plus IL-2 (100 U/ml) and then cultured with IL-4 (10 ng/ml), IL-13 (10 ng/ml) and IL-2 (100 U/ml) for 5 days. All cytokines were from R&D Systems. Anti-CD3 (145-2C11), anti-CD28 (37.51), anti-IFN-γ (XMG1.2), anti-IL-12 (C17.8) and anti-IL-4 (11B11) mAb were from eBioscience. Cells were cultured in RPMI-1640 Medium, 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml of Penicillin/Streptomycin, and 0.05 mM 2-mercaptoethanol.
7
Th1 Polarization of 5CC7 and OT-II T Cells
8
Intracellular Staining and Flow Cytometry
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For measurement of transcription factors, cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions and then stained with monoclonal antibodies specific for FoxP3 (FJK-16s; eBioscience), Ki67 (SolA15; eBioscience), and HIF1α (241812; R&D Systems). For intracellular staining for cytokines, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of GolgiStop (BD Biosciences) in complete media for 4 h at 37°C. After surface marker staining, cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) and then stained with anti-IFNγ (XMG1.2; eBioscience), anti–IL-4 (11B11; eBioscience), anti–IL-5 (TRFK5), and anti–IL-13 (eBio13A; eBioscience) antibodies. Antibodies were from BioLegend unless otherwise stated. An LSR Fortessa (BD Biosciences) was used for multiparameter analysis, and data were analyzed with FlowJo software (TreeStar).
9
Measuring Cytokine Responses to αGal-Cer
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Mice were injected intravenously with either 2 μg αGal-Cer (Abcam) or PBS, and harvested 2 h later for detection of IL-4 and IFNγ by flow cytometry and serum IL-4. For serum detection of IFNγ, mice were harvested 16 h following αGal-Cer injection. Anti-IL-4 (11B11, eBioscience, cat. no.: 17-7041-82, 1:100) and anti-IFNγ (XMG1.2, BioLegend, cat. no.: 505813, 1:100) antibodies were used in conjunction with a Cytofix/Cytoperm Kit (Becton Dickinson).
10
Th17 Cell Differentiation Protocol
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