The in vitro differentiated CD4+ T cells were stimulated using an immobilized anti-TCR-β mAb (3 µg/ml, H57–597, BioLegend) for 6 h in the presence of monensin (2 µM). The cells were fixed with 4% paraformaldehyde (Cat#163–20145, Wako) and permeabilized with permeabilization buffer (50 mM NaCl, 5 mM EDTA, 0.02% NaN3, and 0.5% Triton X-100). Then, the cells were stained using the following antibodies, anti-IL-4-PE (Cat#504103, BioLegend, 1:50), anti-IL-5-APC (Cat#504305, BioLegend, 1:50), anti-IL-13-PE (Cat#12–7133–41, eBiosience, 1:50), anti-IFN-γ-FITC (Cat#562019, BD Biosciences, 1:500), and anti-IL-2-APC (Cat#503809, BioLegend, 1:50). For the intracellular staining of Gata3, the Foxp3/Transcription Factor Staining Buffer Kit (cat#TNB-0607, TONBO) was used according to the manufacturer’s protocol. Flow cytometry was performed using a Gallios Flow Cytometer instrument (Beckman Coulter) and a FACS Caliber instrument (BD Biosciences) and the results were analysed using the FlowJo software program (Tree Star, Ashland, OR, USA).
Anti tcr β mab
Manufactured by BioLegend
Anti-TCR-β mAb is a monoclonal antibody that binds to the beta chain of the T cell receptor (TCR) complex. It is designed for use in flow cytometry and other immunological applications to identify and study T cells.
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Lab products found in correlation
Anti il 5 apc, by BioLegend (1 mentions)
Anti il 2 apc, by BioLegend (1 mentions)
Facs caliber instrument, by BD (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Anti il4 pe, by BioLegend (1 mentions)
Streptavidin horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ mab, by BD (1 mentions)
Mouse il 13 duoset elisa, by R&D Systems (1 mentions)
Tmb peroxidase eia substrate kit, by Bio-Rad (1 mentions)
Anti il 4 mab, by BD (1 mentions)
Anti il 2 mab, by BD (1 mentions)
Cd4 cd62l t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Sodium pyruvate, by Fujifilm (1 mentions)
Sodium pyruvate, by Nacalai Tesque (1 mentions)
2 mercaptoethanol, by Fujifilm (1 mentions)
Mem non essential amino acids, by Nacalai Tesque (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Naive cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
4 protocols using anti tcr β mab
1
Intracellular Cytokine Staining of CD4+ T Cells
2
Cytokine Production by Activated T Cells
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The CD4+ T cells cultured for five days were stimulated using an immobilized anti-TCR-β mAb (3 µg/mL, H57–597, BioLegend) for 16 h, and the culture supernatants were recovered. The amount of cytokines in the recovered supernatants was determined with ELISA using the following antibodies and reagents: anti-IL-4 mAb (Cat#554387, BD Biosciences), biotin-anti-IL-4 mAb (Cat#554390, BD Biosciences), anti-IL-5 mAb (Cat#554393, BD Biosciences), biotin-anti-IL-5 mAb (Cat#554397, BD Biosciences), anti-IFN-γ mAb (Cat#551216, BD Biosciences), biotin-anti-IFN-γ mAb (Cat#554410, BD Biosciences), anti-IL-2 mAb (Cat#554424, BD Biosciences), biotin-anti-IL-2 mAb (Cat#554426, BD Biosciences), streptavidin horseradish peroxidase (Cat#434323, Invitrogen), and TMB peroxidase EIA substrate kit (Cat#1721066, BioRad). For IL-13, the mouse IL-13 Duoset ELISA (Cat#DY413, R&D systems) was used for detecting the levels of the IL-13 cytokine. All antibodies and reagents were used according to the manufacturer’s protocols.
3
Naive CD4 T Cell Differentiation
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Naive CD4 T (CD44lowCD62LhighCD25negative) cells were prepared using a CD4+CD62L+ T cell isolation kit II (cat#130-093-227; Miltenyi Biotec). Naive CD4 T cells (1.5 × 106) were stimulated with an immobilized anti-TCR-β mAb (3 μg ml−1, H57-597; BioLegend) and an anti-CD28 mAb (1 μg ml−1, 37.5; BioLegend) for 2 days under the conditions indicated. Next, the cells were transferred to a new plate and further cultured in the presence of cytokines. The cytokine conditions were as follows: IL-2 conditions, IL-2 (10 ng ml−1); Neutral (Thn) conditions, IL-2 (10 ng ml−1), anti-IL-4 mAb (5 μg ml−1, 11B11; BioLegend) and anti-IFN-γ mAb (5 μg ml−1, R4-6A2; BioLegend); Th2 conditions, IL-2 (10 ng ml−1), IL-4 (1 ng ml−1), and anti-IFN-γ mAb (5 μg ml−1, R4-6A2; BioLegend).
4
Naive CD8 T Cell Activation and Glutamine Deprivation
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Naive CD8 T (CD44lowCD62Lhigh) cells were prepared using a Naive CD8+ T-cell Isolation kit (cat#130-096-543; Miltenyi Biotec, San Diego, CA, USA). Naive CD8 T cells (1.5 × 106) were stimulated with immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) and anti-CD28 mAb (1 μg/ml, 37.5; BioLegend) for 2 days in the presence of IL-2 (10 ng/ml, Pepro Tech). The cells were then transferred to a new plate and further cultured in the presence of IL-2 (10 ng/ml). The cells were cultured in RPMI 1640 with l -glutamine (cat#189-02025; Wako Chemicals) supplemented with 10% fetal bovine serum, 2 mM l -glutamine (16948-04; Nacalai Tesque, Kyoto, Japan), 1 mM sodium pyruvate (cat#06977-34; Nacalai Tesque), 1% MEM nonessential amino acids (cat#06344-56; Nacalai Tesque), 10 mM HEPES (cat#15630-080; Thermo Fisher Scientific, Waktham, MA, USA), 55 μM 2-Mercaptoethanol (cat#21985-023; Thermo Fisher Scientific), and 1% penicillin-streptomycin (cat#26253-84; Nacalai Tesque). For glutamine-deprived conditions, the cells were cultured in RPMI 1640 without l -glutamine (cat#183-02165; Wako Chemicals) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 10 mM HEPES, 55 μM 2-Mercaptoethanol, and 1% penicillin-streptomycin.
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