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Anti cd3e 145 2c11

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Anti-CD3e (145-2C11) is a monoclonal antibody that binds to the CD3e subunit of the T cell receptor complex. It is commonly used for in vitro studies of T cell activation and function.

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Lab products found in correlation

Anti ifn γ xmg1, by BioLegend (2 mentions) Anti il 4 11b11, by BioLegend (2 mentions) Anti cd28 37, by BioLegend (2 mentions) Tgf β, by R&D Systems (2 mentions) Anti cd4 gk1, by BioLegend (2 mentions) Anti cd8 53 6.7, by BioLegend (2 mentions) Anti cd45 30 f11, by BioLegend (1 mentions) Fix perm kit, by BD (1 mentions) Anti b220 ra3 6b2, by Thermo Fisher Scientific (1 mentions) Zombie fixable viability kit, by BioLegend (1 mentions) Anti fcγiii 2 receptor antibody, by BD (1 mentions) Anti pd 1 29f 1a12, by BioLegend (1 mentions) Anti ifn γ xmg1.2, by BD (1 mentions) Golgistop, by BD (1 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Anti cd25 pc61, by Thermo Fisher Scientific (1 mentions) Anti cd4 rm4 5, by Thermo Fisher Scientific (1 mentions) Anti α sma epr5368, by Abcam (1 mentions) Anti cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Anti cd31 390, by Thermo Fisher Scientific (1 mentions)

6 protocols using anti cd3e 145 2c11

1

Generation of Murine Regulatory T Cells

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CD4+CD44−CD62L+ naive T cells were cultured with 3 mg/mL plate-bound anti-CD3e (145-2C11, BioLegend) in the presence of 1 mg/mL anti-CD28 (37.51, BioLegend), 3 ng/mL TGF-β (R&D Systems), 10 ng/mL IL-2, 5 mg/mL anti-IL-4 (11B11, BioLegend), and 5 mg/mL anti-IFN-γ (XMG1.2, BioLegend) for 5 days. Cells were then rested in the larger cell-culture plate for another 3 days and subjected to the analysis.
Tanaka S., Pfleger C., Lai J.F., Roan F., Sun S.C, & Ziegler S.F. (2018). KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners. Cell reports, 23(3), 796-807.
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2

Immune Cell Profiling in Draining Lymph Nodes

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Draining lymph nodes (dLNs) were harvested and single-cell suspensions were prepared. For the exclusion of dead cells, the Zombie fixable viability kit (BioLegend) was used. Cells were incubated in FACS staining buffer (PBS containing 1% BSA and 5 mM EDTA) with anti-FcγIII/II receptor antibody (BD), and anti-CD45 (30-F11, BioLegend), anti-CD4 (Gk1.5, BioLegend), anti-CD8 (53–6.7, BioLegend), anti-CD3e (145-2C11, BioLegend), anti-B220 (RA3-6B2, eBioscience), and anti-PD-1 (29F.1A12, BioLegend) antibodies. For intracellular IFN-γ and Gzm B staining, cells were stimulated with 25 ng/ml PMA and 1 µg/ml Ionomycin in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete RPMI) with monensin (Golgi Stop, BD). After five hours of incubation, cell surface staining was followed by intracellular cytokine staining using the Fix/Perm Kit (BD) in accordance with the manufacturer’s instructions with anti–IFN-γ (XMG1.2, BD) and anti-Gzm B (NGZB, eBioscience) antibodies. Fluorescence-minus-one controls were used as negative controls. Cells were acquired on the Gallios (Beckman-Coulter) and data were analyzed using the FlowJo software (v7.6.5).
Tanaka R., Ichimura Y., Kubota N., Saito A., Nakamura Y., Ishitsuka Y., Watanabe R., Fujisawa Y., Kanzaki M., Mizuno S., Takahashi S., Fujimoto M, & Okiyama N. (2020). Activation of CD8 T cells accelerates anti-PD-1 antibody-induced psoriasis-like dermatitis through IL-6. Communications Biology, 3, 571.
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3

Generation of Murine Regulatory T Cells

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CD4+CD44−CD62L+ naive T cells were cultured with 3 mg/mL plate-bound anti-CD3e (145-2C11, BioLegend) in the presence of 1 mg/mL anti-CD28 (37.51, BioLegend), 3 ng/mL TGF-β (R&D Systems), 10 ng/mL IL-2, 5 mg/mL anti-IL-4 (11B11, BioLegend), and 5 mg/mL anti-IFN-γ (XMG1.2, BioLegend) for 5 days. Cells were then rested in the larger cell-culture plate for another 3 days and subjected to the analysis.
Tanaka S., Pfleger C., Lai J.F., Roan F., Sun S.C, & Ziegler S.F. (2018). KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners. Cell reports, 23(3), 796-807.
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4

Multiparametric Flow Cytometry Analysis

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Single cell suspension was obtained as previously described (34 (link)). The cells were stained with antibodies, purchased from either BioLegend [anti-CD3e (145-2C11, RRID: AB_312660), anti-CD8𝛼 (53-6.7, RRID: AB_2888883), anti-CD11b (M1/70, RRID: AB_312791), anti-CD25 (PC61.5, RRID: AB_312847), anti-CD29 (HMβ1-1, RRID: AB_312885), anti-CD31 (390, RRID: AB_10613644), anti-CD45 (30-F11, RRID: AB_2563598), anti-CD140a (PDGFRα, RRID: AB_1953268, APA5), anti-CD324 (EpCAM, G8.8, RRID: AB_1134172), anti-VEGFR3 (AFL4, AB_10680790), and anti-PDPN (8.1.1, RRID: AB_2629802)] or from Thermo Fisher Scientific [anti-CD4 (RM4-5, RRID: AB_464902), anti-CD69 (H1.2F3, RRID: AB_1210795), anti-FoxP3 (FJK-16s, RRID:AB_467575), and anti-NK1.1 (PK136, RRID: AB_2536075)] or from Abcam [anti-αSMA (EPR5368, RRID: AB_11129103)]. Cells were also stained with Fixable Viability Dye (Thermo Fisher Scientific) and dead cells were gated out from the analysis. Cell types were determined as following; lymphatic cells: CD45+/CD3+, CD8 T+ cells: CD45+/CD3+/CD8+, Tregs: CD45+/CD3+/CD4+/FoxP3+, cancer cells (MOC1): CD45−/ CD31−/EpCAM+/CD140a−/VEGFR3−, cancer cells (MC38-luc): CD45−/CD31−/CD29+/αSMA−, CAFs (MOC1): CD45−/CD31−/EpCAM−/CD140a+/VEGFR3−, CAFs (MC38-luc): CD45−/CD31−/αSMA+, vascular endothelial cells: CD45−/CD31+/EpCAM−/CD140a−/VEGFR3−, lymphatic endothelial cells: CD45−/CD31−/EpCAM−/CD140a−/VEGFR3+.
Kato T., Furusawa A., Okada R., Inagaki F., Wakiyama H., Furumoto H., Fukushima H., Okuyama S., Choyke P.L, & Kobayashi H. (2023). Near-Infrared Photoimmunotherapy Targeting Podoplanin-Expressing Cancer Cells and Cancer-Associated Fibroblasts. Molecular cancer therapeutics, 22(1), 75-88.
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5

Fluorescent Antibody-Based Immune Cell Sorting

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Fluorescent-dye-conjugated antibodies were purchased from BD-Pharmingen (USA) (anti-CD45.2, 104; anti-CD45R, RA3–6B2); eBioscience (USA) (anti-CD103, 2E7; anti-MHC II, M5; anti-F4/80, BM8; anti-CD11b, M1/70; anti-CD11c, N418; anti-Siglec F, E50–2440; anti-CD3e,145–2C11; anti-Ly6G, RB6–8C5) or BioLegend (USA) (anti-CD64 X54–5/7.1). Live/Dead staining was performed using Aqua fixable dead cell stain (Invitrogen). Macrophages were sorted as Aqua–CD45+Lin–(CD3–B220–Siglec F–LY6G–) MHCII+F4/80+CD11B+CD11C+CD103–) using a FACS Aria cell sorter flow cytometer (Becton Dickinson).
Matheis F., Muller P.A., Graves C.L., Gabanyi I., Kerner Z.J., Costa-Borges D., Ahrends T., Rosenstiel P, & Mucida D. (2020). Adrenergic signaling in muscularis macrophages limits infection-induced neuronal loss. Cell, 180(1), 64-78.e16.
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6

NK Cell Phenotyping in Lm-Infected Mice

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Spleens were harvested from Lm infected or naïve ADAPwt/wt × NKp46-Crehet and conditional ADAPfl/fl × NKp46-Crehet mice and processed into single cell suspension. For flow cytometric analysis single cell suspensions were treated with anti-CD16/32 (BioLegend). Cell suspensions were furthermore surface stained with a lineage mix (anti-CD3e, 145-2C11; anti-CD4, GK1.5; anti-CD8, 53-6.7; anti-CD19, 6D5; and anti-TER-119, TER-119; all FITC from BioLegend, except anti-TER-119 which was from Thermo Fisher). Furthermore, the following antibodies were used: anti-NK1.1 (APC-Cy7, PK136), anti-CD122 (PE-Cy7, TM-ß1), anti-CD11b (BV510, M1/70), anti-CD18 (AF647, M18/2), anti-CXCR4 (APC, L276F12), anti-CD29 (PE, HMß1-1) all from BioLegend as well as anti-NKp46 (V450, 29A1.4, BD Biosciences), and anti-CD11a (PE, 2D7, BD Pharmingen). Afterwards, cells were analyzed with a BD LSR Fortessa (BD Biosciences). Flow cytometric data were analyzed using the BD FACS Diva v6.1.3 software.
Böning M.A., Trittel S., Riese P., van Ham M., Heyner M., Voss M., Parzmair G.P., Klawonn F., Jeron A., Guzman C.A., Jänsch L., Schraven B., Reinhold A, & Bruder D. (2020). ADAP Promotes Degranulation and Migration of NK Cells Primed During in vivo Listeria monocytogenes Infection in Mice. Frontiers in Immunology, 10, 3144.
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