Anti cd3 clone 2c11

Manufactured by BioXCell

Anti-CD3 (clone 2C11) is a laboratory reagent used for the activation and expansion of T cells. It binds to the CD3 complex on the surface of T cells, a key step in the initiation of T cell activation and proliferation.

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Anti-CD3 (70 citations) Anti-CD3 (clone 145-2C11; (14 citations) Anti-CD3 (2C11; (7 citations) Anti-CD3 antibody (clone 145-2C11, (3 citations) Anti-mouse CD3 (clone 2C11, (2 citations) Anti-CD3 monoclonal antibody (clone 145-2C11, (2 citations)

6 protocols using anti cd3 clone 2c11

CFSE-Based T Cell Activation Assay

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A total of 5 × 10⁶ T cells were loaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen; cat. no. C34554) according to the manufacturer's instructions. Cells were blocked with 50% FBS, washed twice with RPMI and stimulated with 1 μg ml⁻¹ plate-bound anti-CD3 (clone 2C11) plus 1 μg ml⁻¹ anti-CD28 antibodies (clone 37.51; both Bio X Cell) with or without 50 U ml⁻¹ IL-2 (NIH) for 4 days. CFSE dilution was monitored daily using an LSRII flow cytometer and FACSDiva Software (BD Biosciences) and further analysed with FlowJo Software (Tree Star).

Vaeth M., Yang J., Yamashita M., Zee I., Eckstein M., Knosp C., Kaufmann U., Karoly Jani P., Lacruz R.S., Flockerzi V., Kacskovics I., Prakriya M, & Feske S. (2017). ORAI2 modulates store-operated calcium entry and T cell-mediated immunity. Nature Communications, 8, 14714.

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In Vitro Activation of CD4+ T Cells

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All in vitro experiments were conducted in accordance with UCLA’s institutional policy on humane and ethical treatment of animals following protocols approved by the Animal Research Committee. Five- to eight-week-old OT-II TCR transgenic mice (Jackson Labs) were used for all experiments. Cell-culture media was RPMI supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% HEPES buffer, 0.1% μM 2-mercaptoethanol. CD4+ T cells were purified using negative selection enrichment kits (Stem Cell Technologies).
In vitro activation of CD4+ T cells was performed by culturing 1×10⁶ cells/mL in tissue culture-treated 24-well plates that were pre-coated with anti-CD3 (clone 2C11; Bio X Cell) at a concentration of 10 μg/mL plus addition of 2 μg/mL soluble anti-CD28 (clone 37.51; Bio X Cell).

Majedi F.S., Hasani-Sadrabadi M.M., Thauland T.J., Li S., Bouchard L.S, & Butte M.J. (2020). T-cell activation is modulated by the 3D mechanical microenvironment. Biomaterials, 252, 120058.

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Differentiation of Pathogenic and Non-pathogenic Th17 Cells

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CD4⁺ T cells were isolated from the spleen and submandibular, axillar, inguinal, and mesenterial lymph nodes of mice by negative enrichment using a MagniSort mouse CD4⁺ T‐cell enrichment kit (eBioscience) and stimulated with 0.25 μg/ml plate‐bound anti‐CD3 (clone 2C11) and 1 μg/ml anti‐CD28 (clone 37.51; both Bio X Cell) antibodies in the presence of 2 μg/ml anti‐IL‐4 (clone 11B11) and 2 μg/ml anti‐IFN‐γ (clone XMG1.2; both eBioscience). For the differentiation into non‐pathogenic Th17 cells, CD4⁺ T cells were cultured in the presence of 20 ng/ml IL‐6 (Peprotech) and 0.5 ng/ ml hTGFβ1 (PeproTech) for 2 or 3 days. For the differentiation into pathogenic Th17 cells, CD4⁺ T cells were cultured in the presence of 20 ng/ml IL‐6 (Peprotech), 20 ng/ml IL‐1β (Peprotech), and 20 ng/ml IL‐23 (eBioscience) for 2 or 3 days.

Kahlfuss S., Kaufmann U., Concepcion A.R., Noyer L., Raphael D., Vaeth M., Yang J., Pancholi P., Maus M., Muller J., Kozhaya L., Khodadadi‐Jamayran A., Sun Z., Shaw P., Unutmaz D., Stathopulos P.B., Feist C., Cameron S.B., Turvey S.E, & Feske S. (2020). STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells. EMBO Molecular Medicine, 12(8), e11592.

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Isolation and Differentiation of Naive CD4+ T Cells

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For T cell differentiations, naive CD4⁺CD62L⁺ T cells were isolated from spleens by magnetic beads following the manufacturer’s instructions (Miltenyi Biotec), or by flow cytometric sorting of CD4⁺CD25⁻CD26L⁺CD44^lo cells to >98% purity, as previously described (37 (link)). Briefly, cells were cultured in IMDM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10⁻⁵ M 2-ME, and 5% FBS. Th17 and Th1 cells were differentiated in 96-well plates coated with 2 μg/ml anti-CD3 (clone 2C11; BioXcell) and 2 μg/ml anti-CD28 (clone 37.51; BioXcell) in the presence of either 20 ng/ml IL-6, 0.2 ng/ml TGF-β1 (PeproTech), 10 μg/ml anti–IFN-γ (clone XMG1.2; BioXcell), and 5 μg/ml anti–IL-4 (clone 11B11; BioXcell) (Th17 condition) or 2 ng/ml IL-12 (PeproTech) and 5 μg/ml anti–IL-4 (Th1 condition). For T cell proliferation, naive CD4⁺ T cells were loaded with 2.5 μM CFSE (Lifesciences).

Brucklacher-Waldert V., Ferreira C., Innocentin S., Kamdar S., Withers D.R., Kullberg M.C, & Veldhoen M. (2016). Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology. The Journal of Immunology Author Choice, 196(12), 4893-4904.

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T Cell Differentiation Induction

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Naïve T cells were isolated from spleens and LNs of Stim1^fl/flCd4Cre, Stim2^fl/flCd4Cre, Stim1^fl/flStim 2^fl/flCd4 Cre, or Orai1^fl/flCd4Cre mice and stimulated with 1 μg/ml plate‐bound anti‐CD3 (clone 2C11 obtained from BioXCell) and 1 μg/ml anti‐CD28 (clone 37.51,obtained from BioXCell) for 3 days in the presence of the following cytokine cocktails for differentiation into T helper (Th) cells. Th1: 10 ng/ml IL‐12 (PeproTech) and 2 μg/ml anti‐IL‐4 (eBioscience), Th17: 20 ng/ml IL‐6 (PeproTech), 0.5 ng/ml human TGF‐β1 (PeproTech), 2 μg/ml anti‐IL‐4 and 2 μg/ml anti‐IFNγ (eBioscience), or iTreg: 2.5 ng/ml TGF‐β1 (PeproTech) in IMDM medium (Cellgro) containing 2 mM L‐glutamine, 50 μM 2‐Mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS.

Letizia M., Wang Y., Kaufmann U., Gerbeth L., Sand A., Brunkhorst M., Weidner P., Ziegler J.F., Böttcher C., Schlickeiser S., Fernández C., Yamashita M., Stauderman K., Sun K., Kunkel D., Prakriya M., Sanders A., Siegmund B., Feske S, & Weidinger C. (2022). Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease. EMBO Molecular Medicine, 14(9), e15687.

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Immune Cell Activation Pathway Profiling

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Anti-CD3 clone 2C-11, anti-CD28 clone PV1, and LFA-1 blocking anti-CD11a clone M17/4 were obtained from BioXCell. Anti-pTyr clone PY-20 and anti-p85 (ABS234) was from Upsate (Millipore). Anti-HEF1 (CasL) clone 2G9 and anti-Pyk2 clone YE353 were obtained from Abcam. Anti-Rac1 (610650) and anti-AKT (559028) were from BD. Anti-pAKT (4051), anti-pERK (9101), and anti-c-Cbl (2747) were from Cell Signaling. Anti-Cdc42 (SC-87), anti-CrkL (SC-319), and anti-Cbl-b (SC-8006) were from Santa Cruz. Secondary antibodies conjugated to appropriate fluorophores were obtained from Molecular Probes and Jackson Immunoresearch. AlexaFluor-conjugated phalloidin was purchased from Molecular Probes. Recombinant mouse ICAM-1-Fc, SDF-1α, and P-selectin were purchased from R&D Systems. The ROCK inhibitor Y-27632, the pan PI3K inhibitor LY-294002, and the PI3Kδ inhibitor IC87114 were obtained from CalBiochem. The myosin inhibitor (s)-nitro-blebbistatin was purchased from Cayman Chemicals. The actin destabilizing drug latrunculin B, the Src inhibitor PP2, the Abl kinase inhibitor STI-571, and the PI3Kγ inhibitor CZC24832 were purchased from Sigma.

Roy N.H., MacKay J.L., Robertson T.F., Hammer D.A, & Burkhardt J.K. (2018). Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1. Science signaling, 11(560), eaat3178.

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