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Cd3e clone 145 2c11

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CD3e (clone 145-2C11) is a monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is commonly used in flow cytometry and other immunological applications to identify and characterize T cells.

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Lab products found in correlation

Cd4 clone gk1, by BioLegend (4 mentions) Cd45 clone 30 f11, by BioLegend (4 mentions) Anti il 17a clone tc11 18h10, by BioLegend (2 mentions) Anti ifn γ clone xmg1, by BioLegend (2 mentions) Fc block, by BioLegend (2 mentions) Zombie violet, by BioLegend (2 mentions) Cd3 clone 17a2, by BioLegend (2 mentions) Cd11a clone i21 7, by BioLegend (2 mentions) Thy1.2 clone 53 2 1, by BioLegend (2 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions) F4 80 clone ci a3 1, by Bio-Rad (2 mentions) Mhc 2 clone m5 114, by Thermo Fisher Scientific (2 mentions) Gr 1 clone rb6 8c5, by BioLegend (2 mentions) Cd45 clone 30 f11, by Thermo Fisher Scientific (2 mentions) Cd11b clone m1 70, by Thermo Fisher Scientific (2 mentions) Cd115 clone afs98, by Thermo Fisher Scientific (2 mentions) Lsrii fortessa, by BD (2 mentions) Cd11c clone n418, by BioLegend (2 mentions) Cd11b clone m1 70, by BioLegend (2 mentions) Cd8α clone 53 6, by BioLegend (1 mentions)

9 protocols using cd3e clone 145 2c11

1

Multiparametric Immune Phenotyping of Cells

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Single cell suspensions were treated with Zombie violet as per the manufacturers insert instructions (Biolegend). Following treatment with Fc block (1 μg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145– 2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4–4), CD4 (clone RM 4–5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11–18H10.1) antibodies (BioLegend). Data was acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the supplementary data.
O’Hara J.M., Redhu N.S., Cheung E., Robertson N.G., Patik I., Sayed S.E., Thompson C.M., Herd M., Lucas K.B., Conaway E., Morton C.C., Farber D.L., Malley R, & Horwitz B.H. (2019). Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization. Mucosal Immunology, 13(1), 172-182.
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2

Splenocyte Activation via Anti-CD3/CD28

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Mouse splenocytes were plated on flat bottom 6-well plates (5×106 per well) precoated overnight with 5 µg/mL of anti-CD3 antibodies (CD3e, Clone 145–2 C11, BioLegend) and incubated in the presence of 3 µg/mL of anti-CD28 antibodies (Clone 37.51, BioLegend) for 3 days. Anti-CD3 and anti-CD28 antibodies were omitted in the control unstimulated group. Cells were counted manually or using flow cytometry with CountBright cell counting beads (Thermo Fisher). Human cells were processed in the same way, except human-specific anti-CD3 (Clone UCHT1, BD Biosciences, precoating with 5 µg/mL overnight) and anti-CD28 (Clone CD28.2, BD Biosciences, 5 µg/mL) antibodies were used.
Kumar A., Ramani V., Bharti V., de Lima Bellan D., Saleh N., Uzhachenko R., Shen C., Arteaga C., Richmond A., Reddy S.M, & Vilgelm A. (2023). Dendritic cell therapy augments antitumor immunity triggered by CDK4/6 inhibition and immune checkpoint blockade by unleashing systemic CD4 T-cell responses. Journal for Immunotherapy of Cancer, 11(5), e006019.
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3

Multiparametric Flow Cytometry Analysis

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Fluorochrome-conjugated anti-CD45.2 (clone 104), CD45.1 (A20), TCR-β (clone H57–597), TCRγδ (clone GL3), NK1.1 (clone PK136), CD44 (clone IM7), CD24 (clone M1/69), Gr1 (clone RB6–8C5), CD11b (clone M170), CD11c (clone N418), F4/80 (clone BM8), B220 (clone RA3–6B2), TER119/Erythroid Cells (clone TER-119), and CD3e (clone 145–2C11) were purchased from Biolegend. PE- or APC-conjugated 5-OP-RU loaded MR1 tetramer was kindly provided by the NIH tetramer facility. Cell death was identified using the Live/Dead Fixable Violet Dead Cell Stain (Invitrogen) or 7-AAD.
Pan Y., Deng W., Xie J., Zhang S., Wan E.C., Li L., Tao H., Hu Z., Chen Y., Ma L., Gao J, & Zhong X.P. (2019). Graded diacylglycerol kinases α and ζ activities ensure mucosal-associated invariant T cell development. European journal of immunology, 50(2), 192-204.
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4

Isolation and Characterization of Microglia

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Mice were deeply anesthetized with a lethal dose of choral hydrate and transcardially perfused with 0.1 M PBS. Brain was removed and one forebrain hemisphere (without olfactory bulb) was minced, incubated in phenol-red free DMEM supplemented with 2% heat inactivated FBS, 10mM HEPES and Collagenase type IV (0.4 mg/mL) for 15 min and then passed through a 19G blunt syringe to obtain a homogeneous cell suspension. Mononuclear cells were separated with a 40% Percoll gradient. Isolated cells were surface stained in FACS buffer for 20–30 min on ice with the following antibodies: CD11b (clone M1/70, eBioscience), F4/80 (clone CI: A3-1, BioRad), CD45 (clone 30F11, eBioscience), MHC II (clone M5/114.15.2, eBioscience), CD3e (clone 145-2C11, Biolegend), Gr-1 (clone RB6–8C5, Biolegend), CD115 (clone AFS98, eBioscience). Multiparameter analysis was performed on a LSR II Fortessa (BD) and analyzed with FlowJo software (Tree Star) (Details are included in the Flow Cytometry Reporting Summary). Dead cells and doublets were excluded from all analysis.
Menard C., Pfau M.L., Hodes G.E., Kana V., Wang V.X., Bouchard S., Takahashi A., Flanigan M.E., Aleyasin H., LeClair K.B., Janssen W.G., Labonté B., Parise E.M., Lorsch Z.S., Golden S.A., Heshmati M., Tamminga C., Turecki G., Campbell M., Fayad Z., Tang C.Y., Merad M, & Russo S.J. (2017). Social stress induces neurovascular pathology promoting depression. Nature neuroscience, 20(12), 1752-1760.
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5

Isolation and Characterization of Microglia

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Mice were deeply anesthetized with a lethal dose of choral hydrate and transcardially perfused with 0.1 M PBS. Brain was removed and one forebrain hemisphere (without olfactory bulb) was minced, incubated in phenol-red free DMEM supplemented with 2% heat inactivated FBS, 10mM HEPES and Collagenase type IV (0.4 mg/mL) for 15 min and then passed through a 19G blunt syringe to obtain a homogeneous cell suspension. Mononuclear cells were separated with a 40% Percoll gradient. Isolated cells were surface stained in FACS buffer for 20–30 min on ice with the following antibodies: CD11b (clone M1/70, eBioscience), F4/80 (clone CI: A3-1, BioRad), CD45 (clone 30F11, eBioscience), MHC II (clone M5/114.15.2, eBioscience), CD3e (clone 145-2C11, Biolegend), Gr-1 (clone RB6–8C5, Biolegend), CD115 (clone AFS98, eBioscience). Multiparameter analysis was performed on a LSR II Fortessa (BD) and analyzed with FlowJo software (Tree Star) (Details are included in the Flow Cytometry Reporting Summary). Dead cells and doublets were excluded from all analysis.
Menard C., Pfau M.L., Hodes G.E., Kana V., Wang V.X., Bouchard S., Takahashi A., Flanigan M.E., Aleyasin H., LeClair K.B., Janssen W.G., Labonté B., Parise E.M., Lorsch Z.S., Golden S.A., Heshmati M., Tamminga C., Turecki G., Campbell M., Fayad Z., Tang C.Y., Merad M, & Russo S.J. (2017). Social stress induces neurovascular pathology promoting depression. Nature neuroscience, 20(12), 1752-1760.
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6

Multicolor Flow Cytometry Analysis

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Single-cell suspensions from spleens, dLNs, non-dLNs, or tumor tissues were prepared as previously described (42 (link)). Combinations of the following fluorochrome-conjugated antibody were used for cell surface or intracellular staining to define populations of NK, CD8, and subsets of CD4 T cells: CD3e (clone 145-2c11), CD8a (clone 53-6.7), CD4 (clone GK1.5), NK1.1 (clone PK136), NKG2D (clone CX5), CD44 (clone eBio4B10), CD11c (clone N418), MHCII (clone M5/114.15.2), CD80 (clone 16-10A1), CD86 (clone PO3), CD40 (clone 1C10), and CD3ζ (clone 6B10.2, BioLegend). For ex vivo restimulation, freshly isolated single-cell suspension was cultured in complete RPMI 1640 medium containing PMA (50 ng/ml) and ionomycin (500 ng/ml) for 6 hours before it was analyzed for IFN-γ production by intracellular staining with an antibody specific to IFN-γ (XMG1.2). Multicolored flow cytometry analyses were performed on LSR II (BD). Data were analyzed with FlowJo software (Tree Star).
Zhang J., Liu D., Li G., Staveley-O’Carroll K.F., Graff J.N., Li Z, & Wu J.D. (2017). Antibody-mediated neutralization of soluble MIC significantly enhances CTLA4 blockade therapy. Science Advances, 3(5), e1602133.
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7

Dissociation and Staining of Murine Tumors

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After in vivo experiments, mouse tumors were rapidly excised and mechanically dissociated in PBS using scissors. The tumors were then digested in serum-free RPMI supplemented with 20 mg/ml DNase I (Roche), 20 mg/ml Dispase II (Roche) and 20 mg/ml collagenase I (Sigma) for 30–60 min at 37 °C with rotation to promote dissociation. The resulting single-cell suspensions were passed through 70 μM strainers twice, and red blood cells in the tumor samples were lysed with red blood cell lysis buffer (eBioscience, #00-4333-57) for 5 min at room temperature. Then, the single-cell suspensions were washed in Cell Staining Buffer (BioLegend) and incubated with the indicated flow antibodies at 4 °C for 30 min. Prior to staining with antibody panels, cells were blocked with a monoclonal antibody against CD16/32 (BioLegend) for 15 min at 4 °C. All the antibodies and reagents used for flow cytometry included ZombieRED (ECD, Biolegend, #423110), CD45 (clone 30-F11, Biolegend, #103116), CD3e (clone 145-2C11, Biolegend, #100328), CD4 (clone RM4-5, Biolegend, #100536), CD8 (clone 53–6.7, Biolegend, #100706), GZMB (clone QA16A02, Biolegend, #372208), PRF1 (clone S16009B, Biolegend, #154404), F4/80 (clone BM8, Biolegend, #123127), CD11b (clone M1/70, Biolegend, #101206), CD86 (clone GL-1, Biolegend, #105006) and CD206 (clone C068C2, Biolegend, #141719).
Zhao Y.X., Wang H., Zhang S.W., Zhang W.X., Jiang Y.Z, & Shao Z.M. (2024). Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy. Cancer Cell International, 24, 131.
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8

Multicolor Flow Cytometry of Immune Cells

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The antibiotics vancomycin, polymyxin B, ampicillin, and neomycin were obtained from Gold Biotechnology, Inc. (St. Louis, MO), and metronidazole was from Sigma-Aldrich Corp. (St. Louis, MO). Liposomal clodronate and control liposomes were obtained from Encapsula NanoSciences LLC (Brentwood, TN). The following reagents and anti-mouse antibodies were used for flow cytometry: Zombie VioletTM fixable viability kit (BioLegend, San Diego, CA), CD45 (clone 30-F11, BioLegend), CD11b (clone M1/70, BioLegend), CD11c (clone N418, BioLegend), CD103 (clone 2E7, eBioscience/ Thermo Fisher Scientific, Waltham, MA), CD64 (clone X54-5/7.1, BioLegend), I-A/I-E (MHCII, clone M5/114.15.2, BioLegend), Ly6C (clone HK1.4, BioLegend), CD3e (clone 145–2 C11, BioLegend), CD4 (clone GK1.5, BioLegend), FoxP3 (clone FJK-16s, eBioscience), IFNγ (clone XMG1.2, BioLegend), IL17A (clone TC11-18H10.1, BioLegend), CD127 (clone A7R34, BioLegend), CD90.2 (Thy1.2, clone 30-H12, BioLegend), and RORγt (clone B2D, eBioscience).
Redhu N.S., Bakthavatchalu V., Conaway E.A., Shouval D.S., Tsou A., Goettel J.A., Biswas A., Wang C., Field M., Muller W., Bleich A., Li N., Gerber G.K., Bry L., Fox J.G., Snapper S.B, & Horwitz B.H. (2017). Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor. eLife, 6, e27652.
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9

Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were treated with Zombie violet as per the manufacturer's insert instructions (Biolegend). Following treatment with Fc block (1 µg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145-2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4-4), CD4 (clone RM 4-5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11-18H10.1) antibodies (BioLegend). The data were acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the Supplementary Data.
O’Hara J.M., Redhu N.S., Cheung E., Robertson N.G., Patik I., Sayed S.E., Thompson C.M., Herd M., Lucas K.B., Conaway E., Morton C.C., Farber D.L., Malley R, & Horwitz B.H. (2019). Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization. Mucosal Immunology, 13(1), 172-182.
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