γδ T cells were sorted from pLNs. CD4+, CD8+ and CD19+ cells were depleted using Dynabeads (Invitrogen) before a negative sorting using Aria III cytometer (BD Biosciences). Highly purity of γδ T cells with untouched TCR was obtained. γδ T cells were cultured in RPMI 1640+Glutamax (Gibco) with 10% FCS, 100 U ml-1 penicillin and streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, non-essential amino acids, 50 μM 2-mercaptoethanol in 96 well plates at 37°C, 5% CO2. When indicated 15–30 U ml-1 of rIL-2 and 15 μg ml-1 rIL-7 (R&D Systems) were used. Cells were cultured on plate-bound with 0.1 μg ml-1 anti-CD3ε (145-2C11) and 10 μg ml-1 anti-CD28 (37.51) (both from eBioscience).
Anti cd3ε 145 2c11
Manufactured by Thermo Fisher Scientific
The Anti-CD3ε (145-2C11) is a monoclonal antibody that binds to the CD3 epsilon subunit of the T cell receptor complex. It is commonly used in research applications to activate T cells.
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Lab products found in correlation
Anti cd28 37.51, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Ril 2, by R&D Systems (1 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions)
Ril 7, by R&D Systems (1 mentions)
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Anti p eif2a, by Cell Signaling Technology (1 mentions)
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Anti bip, by Cell Signaling Technology (1 mentions)
Anti ptbk1 d52c2, by Cell Signaling Technology (1 mentions)
Anti tbk1 d1b4, by Cell Signaling Technology (1 mentions)
Anti pirf3 4d4g, by Cell Signaling Technology (1 mentions)
Anti chop l63f7, by Cell Signaling Technology (1 mentions)
Anti sting d2p2f, by Cell Signaling Technology (1 mentions)
Anti irf3 d83b9, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3 5a1e, by Cell Signaling Technology (1 mentions)
2 protocols using anti cd3ε 145 2c11
1
Isolation and Culture of Murine γδ T Cells
2
Antibodies and Flow Cytometry Techniques
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The following antibodies were used for flow cytometry: anti-CD8α (53–6.7), anti-CD4 (L3T4), anti-CD3ε (145-2C11), anti-CD25 (PC61.5), anti-CD69 (H1.2F3), anti-CD44 (IM7), anti-CD62L (MEL-14), CD19 (1D3), CD45 (30-F11), and Ly6G (1A8) all from BD Biosciences or BioLegend. The splenocytes were stained with the indicated panel of antibodies according to the manufacturers’ instructors, and then analyzed by BD FACSCalibur or BD LSR II FACs machines. Purified anti-CD3ε (145-2C11) and anti-CD28 (37.51; both from eBioscience) were used at the appropriate concentration for T cell activation. The following antibodies were used for immunoblotting analysis: anti-STING (D2P2F; Cell Signaling), anti-TBK1 (D1B4; Cell Signaling), anti-pTBK1 (D52C2; Cell Signaling), anti-IRF3 (D83B9, Cell Signaling), anti-pIRF3 (4D4G, Cell Signaling), anti-cleaved PARP (D64E10; Cell Signaling), anti-cleaved caspase 3 (5A1E; Cell Signaling), anti-Bip (C50B12; Cell Signaling), anti-Chop (L63F7; Cell Signaling), anti-eIF2a (D7D3; Cell Signaling), and anti-p-eIF2a (119A11; Cell Signaling).
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