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Anti cd3ε

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Anti-CD3ε is a lab equipment product designed for use in research applications. It serves as a primary reagent for the activation of T cells. The product targets the CD3ε subunit of the T cell receptor complex, which is essential for T cell activation and signal transduction.

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Lab products found in correlation

Anti cd28, by BioXCell (5 mentions) Anti ifn γ, by BioXCell (3 mentions) Anti il 4, by BioXCell (3 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Ic fixation buffer, by Thermo Fisher Scientific (2 mentions) Rhtgf β1, by Thermo Fisher Scientific (2 mentions) Anti cd28, by Thermo Fisher Scientific (2 mentions) Oligomycin, by Merck Group (2 mentions) Rotenone, by Agilent Technologies (2 mentions) Antimycin a, by Agilent Technologies (2 mentions) Streptomycin, by BioXCell (2 mentions) Rmil 6, by BioLegend (2 mentions) Rmil 23, by BioLegend (2 mentions) Mouse naive cd4 isolation kit, by STEMCELL (2 mentions) Rapamycin, by LC Laboratories (2 mentions) Transforming growth factor β1, by Thermo Fisher Scientific (1 mentions) 6 formylindolo 3 2 b carbazole ficz, by Enzo Life Sciences (1 mentions) Anti il 2, by BioXCell (1 mentions) Jes6 1a12, by BioXCell (1 mentions) Lyse fix buffer, by BD (1 mentions)

Spelling variants of this product

Anti-CD3ε (145-2C11, (5 citations) Anti-CD3ε mAb (clone 145-2C11, (3 citations) Anti-CD3ε (2C11; (3 citations) Anti-CD3ε (clone 145-2C11) (3 citations) Hamster anti-CD3ε 2C11 F(ab′)2 fragment (2 citations) Monoclonal anti-mouse CD3ε [145–2c11] (2 citations)

13 protocols using anti cd3ε

1

Th17 Polarization of Splenic Naive CD4+ T Cells

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Splenic naïve CD4+ T cells (2.5 × 104) from SFB young or middle-aged K/BxN mice were enriched by fluorescence-activated cell sorting (FACS Aria) and in vitro cultured in 96-well plates for 4 days in Th17 polarization conditions: anti-CD3ε (plate-coated, 2 μg/ml), anti-CD28 (2 μg/ml), anti-IL-2 (10 μg/ml, JES6-1A12, BioXCell), IL-6 (50 ng/ml, PeproTech), transforming growth factor (TGF)β1 (1 ng/ml, PeproTech), 6-formylindolo [3,2-b] carbazole (FICZ), (300 nM, Enzo Life Sciences).
Teng F., Felix K.M., Bradley C.P., Naskar D., Ma H., Raslan W.A, & Wu H.J. (2017). The impact of age and gut microbiota on Th17 and Tfh cells in K/BxN autoimmune arthritis. Arthritis Research & Therapy, 19, 188.
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2

Phospho-ERK1/2 Signaling in T Cells

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C57BL/6 wild-type (WT) mice were used for examination of phosphoERK1/2 expression upon stimulation. Cells were isolated with magnetic negative enrichment using CD3 isolation kit (StemCell). CD3 enriched cells were kept ice cold until time of stimulation in 37°C water bath. Cells were first labeled with anti-CD3ε (10μg/ml) and anti-CD28 (5μg/ml) (BioXcell) antibodies in ice cold PBS for 30min on ice. Cells were washed and then stained with cross-linker goat anti-Armenian hamster IgG (Jackson Immuno Research Laboratories) (20μg/ml) and anti-CD44/ anti-CD62L antibodies for additional 30min on ice. Tubes were then placed in water bath to start stimulation and lyse/fix buffer (BD) was added to stop stimulation at indicated times. Cells were permeabilized with Perm III buffer (BD) before being stained for CD3, CD4, CD8, and pERK1/2. Samples were acquired in LSRII cytometer and analyzed using Flowjo software.
Thapa P., Guyer R.S., Yang A.Y., Parks C.A., Brusko T.M., Brusko M., Connors T.J, & Farber D.L. (2021). Infant T cells are developmentally adapted for robust lung immune responses through enhanced T cell receptor signaling. Science immunology, 6(66), eabj0789.
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3

Th17 Cell Differentiation by DCs and Rg3

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Naïve CD4+ T cells (CD44lowCD62LhighCD25−) were isolated from wild-type mice using a cell sorter (BD BioScience, San Jose, CA, USA). For DC-mediated Th17 cell differentiation, BMDCs were co-cultured with naive CD4+ T cells in the presence of anti-CD3ε (0.3 μg/mL) antibody (145-2C11, BioXcell, NH, USA), TGFβ (1.5 ng/mL) (PeproTech, Rocky Hill, NJ, USA), and LPS (100 ng/mL) (Sigma, Seoul, Korea) for 96 h. For DC-free Th17 cell differentiation, anti-CD3ε (1 μg/mL) and anti-CD28 (1 μg/mL) (37.51, BioXcell, West Lebanon, NH, USA) were pre-coated in a 96-well flat-bottom plate overnight at 4 °C. After washing the plate with cold PBS three times, 1 × 105 naïve CD4+ T cells were stimulated with IL-6 (10 ng/mL) and TGFβ (1.5 ng/mL) (PeproTech, Rocky Hill, NJ, USA) for 96 h. For Rg3 (LKT Labs, Saint Paul, MN, USA; dissolved in DMSO) treatment, various concentrations (9.37, 18.75, and 37.5 μg/mL) were added at the beginning of in vitro culture.
Park Y.J., Cho M., Choi G., Na H, & Chung Y. (2020). A Critical Regulation of Th17 Cell Responses and Autoimmune Neuro-Inflammation by Ginsenoside Rg3. Biomolecules, 10(1), 122.
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4

Detailed Immune Cell Phenotypic Analysis

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Cell suspensions were first stained for surface markers (such as CD45.2 and CD8) and then fixed and stained for TCF1 (clone C63C9), T-bet (clone 4B10), Eomes (clone Dan11mag), Blimp1 (clone 5E7), or Ki67 (clone B56) using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) according to manufacturer instructions. To assess IFN-γ and TNF production, live cell suspensions were stimulated by plate bound anti-CD3ε (1 μg/ml coating concentration; 145-2C11; BioXCell) and anti-CD28 (1 μg/ml coating concentration; 37.51; BioXCell) or PMA and Ionomycin (20 ng/ml and 1 μg/ml, respectively, both from Sigma-Aldrich) for 2 hours at 37 °C, after which GolgiPlug was added, and the cells incubated for an additional 4 hours in 37 °C. After staining for surface markers, samples were fixed with IC Fixation Buffer (eBioscience), treated with permeabilization buffer (eBioscience), and stained with fluorescently labeled anti-IFN-γ (clone XMG1.2) and anti-TNF (clone MP6-XT22) antibodies, followed by flow cytometry analysis (BD Biosciences LSRII and Beckman Coulter Cytoflex LX). Details pertaining to antibody staining, including clone, fluorophore, and dilution, are provided in table S2.
Zhang M., Zhao Z., Pritykin Y., Hannum M., Scott A.C., Kuo F., Sanghvi V., Chan T.A., Seshan V., Wendel H.G., Schietinger A., Sadelain M, & Huse M. (2021). The miR-200c–EpCAM axis reprograms therapeutic T cells to enhance anti-tumor immunity in mouse models. Science translational medicine, 13(611), eabg4328.
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5

Differentiation of Naive CD4+ T Cells

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Naive CD4+ T cells were cultured in tissue culture–treated multiwell plates that were coated overnight with 2 μg/mL of anti-CD3ε (Bio X Cell) at 106 cells/mL. Th1, Th2, and Th9 were cultured as previously described (59 (link)), and in some cases IL-6 and/or IL-21 was added to these cultures in varying concentrations as noted. In cytokine screening experiments, cells were cultured in flat-bottom, 96-well plates with plate-bound anti-CD3 and soluble anti-CD28 as per above. Cytokines were added at day 0 of culture (IL-4, 10 ng/mL; IL-6, 20 ng/mL; IL-12, 10 ng/mL; IL-21, 100 ng/mL; Pam3CSK4, 2 μg/mL; TGF-β, 2 ng/mL; TNF 100 ng/mL; see Supplemental Table 3 for other cytokines used), and cells were expanded 1:4 in fresh media with no additional cytokines day 3 of culture. All cytokines were purchased from PeproTech, except TGF-β, which was purchased from R&D Systems, Bio-Techne. Pam3CSK4 was purchased from InvivoGen.
Park S., Griesenauer B., Jiang H., Adom D., Mehrpouya-Bahrami P., Chakravorty S., Kazemian M., Imam T., Srivastava R., Hayes T.A., Pardo J., Janga S.C., Paczesny S., Kaplan M.H, & Olson M.R. (2020). Granzyme A–producing T helper cells are critical for acute graft-versus-host disease. JCI Insight, 5(18), e124465.
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6

Naive CD4 T Cell Differentiation

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Naive CD4 T cells from WT, STAT3C+/+, Foxp3-GFP, Nur77-GFP, and CD45.1 mice were isolated by FACS sorting as described previously (Yeh et al., 2011 (link)) or by magnetic bead enrichment using the mouse naive CD4 isolation kit (Stem Cell Technology) according to the manufacturer’s protocol. Naive CD4 T cells were stimulated in R10 media (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 × nonessential amino acids, 1 μM sodium pyruvate, and 50 μM β-mercaptoethanol) with plate-bound 10 μg/ml anti-CD3ε (BioXCell, clone 145C-11) and soluble 1 μg/ml anti-CD28 (eBioscience, clone 37.51), 20 ng/ml rmIL-6 (Biolegend), 2.5 ng/ml rhTGFβ1 (Peprotech), 10 ng/ml rmIL-23 (Biolegend), 10 μg/ml anti-IL-4 (BioXCell, 11B11), and 10 μg/ml anti-IFNγ (BioXCell, XMG1.2) for indicated amount of time. The following inhibitors were used in this study:
2.5 ng/ml oligomycin (Sigma-Aldrich),
250 nM antimycin A and 250 nM rotenone (Agilent technologies),
50 nM rapamycin (LC Laboratories).
Shin B., Benavides G.A., Geng J., Koralov S.B., Hu H., Darley-Usmar V.M, & Harrington L.E. (2020). Mitochondrial Oxidative Phosphorylation Regulates the Fate Decision between Pathogenic Th17 and Regulatory T Cells. Cell reports, 30(6), 1898-1909.e4.
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7

Measuring CD8+ T Cell Degranulation

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For degranulation assays (30 (link)), activated CTLs were stimulated in plates coated with anti-CD3ε (BioXCell) or mixed at a 1:1 ratio with peptide-pulsed or non-pulsed targets at 37°C in the presence of anti-CD107a-PE-labeled antibody (clone 1D4B, BioLegend). Upon degranulation, CD107a is exposed on the cell surface, allowing binding and subsequent internalization of the anti-CD107a-PE. At indicated time points, plates were placed on ice and cells transferred into cold PBS, stained with anti-CD8α (clone 53–6.7, BioLegend) antibody, and analyzed via flow cytometry. For analysis, the percent of PE+ cells in the CD8+ gate indicates cells that have degranulated, having been exposed to anti-CD107a during the assay.
Kapnick S.M., Stinchcombe J.C., Griffiths G.M, & Schwartzberg P.L. (2017). Inducible T cell kinase regulates the acquisition of cytolytic capacity and degranulation in CD8+ cytotoxic T lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 198(7), 2699-2711.
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8

Intracellular TNFα Staining in Activated Splenocytes

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Total splenocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 100U/ml penicillin-streptomycin (Life Technologies) and 2mM L-glutamine (Life Technologies), and stimulated as follows. For TNFα intracellular staining, 5 × 106 thymocytes or splenocytes were stimulated with or without plate-coated anti-CD3ε (10 μg/ml, 145-2C11, Bio-X-Cell) plus soluble anti-CD28 (1μg/ml, 37.51, Bio-X-Cell) in the presence of Protein Transport Inhibitor Cocktail (eBioscience) for 17 hours at 37°C in 5% CO2. After the incubation, cells were harvested and washed once with 1X PBS, and stained with fixable viability dye before surface staining. Following the surface stain, cells were fixed and permeabilized with IC Fixation Buffer (eBioscience) and then stained for intracellular TNFα (MP6-XT22, eBioscience).
Hsu F.C., Belmonte P., Constans M., Chen M.W., McWilliams D.C., Hiebert S.W, & Shapiro V.S. (2015). HDAC3 is required for T cell maturation. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1578-1590.
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9

Stimulation of Naive and Memory T Cells

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Naïve and memory WT and Crlf2-/- P14 T cells were obtained and purified as described above in ‘Cell transfers and infections’. 1 × 106 CD8+ T cells were plated in 0.75 ml/well in 48 well plates or 2 × 106 CD8+ T cells were plated in 1.5 ml/well total volume in 24 well plates with either medium or 100 ng/ml TSLP (R&D Systems) with or without stimulation. For in vitro stimulation of naïve cells, the plates were pre-coated with 2 μg/ml anti-CD3ε (BioXcell) and 1 μg/ml of soluble anti-CD28 was added. For in vitro stimulation of memory cells, 1 μg/ml of soluble anti-CD3 was added.
Ebina-Shibuya R., West E.E., Spolski R., Li P., Oh J., Kazemian M., Gromer D., Swanson P., Du N., McGavern D.B, & Leonard W.J. (2021). Thymic stromal lymphopoietin limits primary and recall CD8+ T-cell anti-viral responses. eLife, 10, e61912.
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10

T Cell Activation and TGF-beta Modulation

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All cells were cultured in RPMI supplemented with 10% heat-inactivated Fetal Bovine Serum (Atlanta Biologicals, Lawrenceville, GA, USA), 2mM L-glutamine (Mediatech, Manassas, VA, USA), and 1% penicillin/streptomycin (Mediatech, Manassas, VA, USA). CD8 T cells were activated with 100 ng/mL OVA257 peptide (SIINFEKL, New England Peptide, Gardner, MA, USA), 1 µg/mL anti-CD3ε (clone 145-2C11, Bio X Cell, West Lebanon, NH, USA), or 1 µg/mL anti-CD3ε and 5 µg/mL anti-CD28 (clone 37.51, Bio X Cell) for 5 days in media supplemented with 30 U/mL IL-2 (R&D Systems, Minneapolis, MN, USA). When noted, cells were incubated with TGFβ1 (EMD Chemicals, Inc., Gibbstown, NJ, USA) at a final concentration of 20 ng/mL.
O’Sullivan J.A., Kohlhapp F.J., Zloza A., Plaza-Rojas L., Burke B., Dulin N.O, & Guevara-Patiño J.A. (2023). Memory Precursors and Short-Lived Effector T cell Subsets Have Different Sensitivities to TGFβ. International Journal of Molecular Sciences, 24(4), 3930.
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