Cd3 145 2c11

Manufactured by BD

Sourced in United States

The CD3 (145-2C11) is a lab equipment product manufactured by BD. It is a monoclonal antibody that binds to the CD3 complex on the surface of T cells, which is a key component of the T cell receptor complex. The CD3 antibody is commonly used in flow cytometry and cell culture applications to detect and study T cells.

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Anti-CD3 (145-2C11, (37 citations) Anti-CD3 mAb (clone 145-2C11, (10 citations) Anti-mouse CD3 (clone 145-2C11, (7 citations) APC-anti-CD3 (145-2C11; (6 citations) CD3-PE (145-2C11), (6 citations) CD3-APC (145-2C11), (5 citations) Anti-CD3-FITC (145-2C11, (4 citations) PE‐Cy7‐anti‐CD3 (145‐2C11), (3 citations) Anti-mouse CD3 (145-2C11, (3 citations) Anti-CD3 (145-2C11) and anti-CD28 (37.51) (2 citations)

12 protocols using cd3 145 2c11

Multiparametric Analysis of Immune Cells

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Flow cytometry and cell sorting were completed on a FACSAria Fusion instrument (BD) and analyzed using FlowJo analysis software (Tree Star). Surface staining was performed at 4°C in the presence of Fc block (2.4G2) in magnetic-activated cell sorting (MACS) buffer (PBS, 0.5% BSA, 2 mM EDTA). Intracellular IRF8 staining was performed using the Foxp3 staining kit (eBioscience 00-5523-00).
The following antibodies were used: CD19 (1D3), CD135 (A2F10.1), MHCII (M5/114.15.2), CD117 (2B8), B220 (RA3-6B2), CD3 (145-2C11), and CD4 (RM4-5) from BD Biosciences; CD3 (145-2C11), CD4 (GK1.5), and MHCII (M5/114.15.2) from Tonbo Biosciences; TER-119 (TER-119), Ly-6G (1A8), B220 (RA3-6B2), CD24 (M1/69), CD115 (AFS98), XCR1 (ZET), CD19 (6D5), CD8α (53-6.7), CD4 (RM4-5), CD11c (N418), and CD3 (17A2) from Biolegend; CD105 (MJ7/18), Siglec-H (eBio440c), CD3 (17A2), CD8α (53-6.7), CD11c (N418), and IRF8 (V3GYWCH) from eBiosciences; and SA-Qdot 605, CD11c (N418), CD317 (eBio927), CD172a (P84), and TCRβ (H57-597) from Invitrogen.

Liu T.T., Ou F., Belk J.A., Bagadia P., Anderson DA I.I.I., Durai V., Yao W., Satpathy A.T., Murphy T.L, & Murphy K.M. (2023). Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation. Genes & Development, 37(7-8), 291-302.

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Single-Cell Analysis of Immune Populations

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Lymph nodes were processed into single cell suspensions using a 70 μm cell strainer. Corneas were excised and incubated with 1 mg/ml collagenase D (11088866001, Roche, Switzerland) for 1 h at 37°C to obtain single cell suspensions. Cells were incubated with Fc block (CD16/CD32) for 15 min at 4°C, followed by incubation with directly conjugated monoclonal antibodies (mAb) to CD3 (145-2C11), CD4 (GK1.5), CD45 (30-F11), CD11b (M1/70), CD11c (HL3), Gr-1 (RB6-8C5), CD40 (3/23) and CD86 (GL1) (all from BD Biosciences, USA) for 30 min at 4°C. For staining of intracellular transcription factor, cells were incubated with Cytofix/Cytoperm (BD Biosciences, USA) for 20 min at 4°C followed by staining with directly conjugated, T-bet (4B10, eBioscience, UK) and RORγt (Q31-378, BD Biosciences, USA) for 30 min at 4°C. Thereafter, the cells were washed and data acquired with a BD LSRII Flow Cytometer (BD Biosciences, USA). Data analysis was performed using the FlowJo software (Miltenyi Biotec, Germany). For calculation of absolute cell numbers, the percentage cell population was multiplied by total cell count.

Yu T., Schuette F., Christofi M., Forrester J.V., Graham G.J, & Kuffova L. (2022). The atypical chemokine receptor-2 fine-tunes the immune response in herpes stromal keratitis. Frontiers in Immunology, 13, 1054260.

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Comprehensive Multiparameter Flow Cytometry

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The Cyan ADP flow cytometer (Beckman Coulter, Indianapolis IN) was used for all flow samples. Antibodies for these surface markers were used for flow cytometry: CD3 (145-2C11, BD Bioscience Franklin Lakes, NJ, USA), CD4 (RM4-5, BD Bioscience), anti-CEA CAR (Wi2, Immunomedics Morris Plains, NJ, USA), CD11b (M1/17, BD Bioscience), Ly6C (AL-21, BD Bioscience), Ly6G (1AB, BD Bioscience), PD-L1 (MIH5, BD Bioscience), CD62L (MEL-14, BD Bioscience), CCR7 (4B12, BD Bioscience), CD44 (IM7, BD Bioscience). Intracellular FoxP3 staining was performed with Mouse FoxP3 Permeabilization Kit (BD Bioscience). Single stain and isotype controls were used for each experiment. Analysis of acquired flow samples was performed with FlowJo software (Tree Star Inc., Ashland OR).

Katz S., Point G.R., Cunetta M., Thorn M., Guha P., Espat N.J., Boutros C., Hanna N, & Junghans R.P. (2016). Regional CAR-T cell infusions for peritoneal carcinomatosis are superior to systemic delivery. Cancer gene therapy, 23(5), 142-148.

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Hematopoietic Cell Profiling in Mouse Tissues

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Hematopoietic stem and progenitor cell and lineage-positive cell profiling was perform on BM, spleen, and thymus of mice from each genotype. Antibodies (clones) used: CD3 (145-2C11) [BD: #553064], CD4 (H129.19) [BD: #553653], CD8 (53-6.7) [BD: #553033], Gr-1 (RB6-8C5) [BD: #553128], B220 (RA3-6B2) [BD: #553090], Ter119 (Ter119) [BD: #553673], Mac1 (M1/70) [BD: #553311], IL7Rα (A7R34) [eBiosceinces: #12-1271-82], cKit (2B8) [BD: #558163], Sca-1 (E13-161.7) [eBiosceinces: #17-5981-83], CD25 (7D4) [eBiosciences: 13-0252-82], CD44 (IM7) [BD: 559250], CD43 (S7) [BD: 561856], HSA (M1/69) [BD: #553262] and BP-1 (6C3) [BD: #553159], CD4 (GK1.5) [BD: #561830], CD8 (7D4) [eBiosciences: #12-0081-82], B220 (RA3-6B2) [BD: #561880].
Flow cytometric gating strategy is described in Supplementary Fig. 6.

Khattar E., Maung K.Z., Chew C.L., Ghosh A., Mok M.M., Lee P., Zhang J., Chor W.H., Cildir G., Wang C.Q., Mohd-Ismail N.K., Chin D.W., Lee S.C., Yang H., Shin Y.J., Nam D.H., Chen L., Kumar A.P., Deng L.W., Ikawa M., Gunaratne J., Osato M, & Tergaonkar V. (2019). Rap1 regulates hematopoietic stem cell survival and affects oncogenesis and response to chemotherapy. Nature Communications, 10, 5349.

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Bone Marrow and Spleen Cell Isolation

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Bone marrow cells were harvested from femurs and tibiae by crushing the bones with a mortar and pestle. The cell suspension was collected after filtering through 40 μ membrane filter (BD). These cells were used for surface staining after red blood cell (RBC) lysis treatment with Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma). Spleens were crushed between two glass slides and isolated cells were used for surface staining with respective antibodies after RBC lysis. Surface staining was done with B220 (RA3-6132, BD), CD3 (145-2C11, BD), Gr-1 (RB6-8C5, BD), Mac1 (M170, BD), CD45.1 (A20, BD), and CD45.2 (104, BD). After surface staining with respective fluorescent antibodies, polychromatic flow acquisition was performed using a LSR II instrument (BD) at the University of Rochester Flow Cytometry Core. Flow data was analyzed using FlowJo software (version 10.0.7 Treestar, Ashland, OR).

Unnisa Z., Singh K.P., Henry E.C., Donegan C.L., Bennett J.A, & Gasiewicz T.A. (2016). Aryl Hydrocarbon Receptor Deficiency in an Exon 3 Deletion Mouse Model Promotes Hematopoietic Stem Cell Proliferation and Impacts Endosteal Niche Cells. Stem Cells International, 2016, 4536187.

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T Cell Proliferation Assay with Polarized Macrophages

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96 well round bottom plates were coated with 1 μg/ml CD3 (145–2C11, BD Biosciences) and 0.5 μg/ml CD28 (37.5, BD Biosciences). Macrophages were M1 (250 ng/ml LPS) or M2 (20 ng/ml IL-4) polarized prior to the experiment for 16 h. CD4⁺CD25⁻ T cells were stained with CFSE proliferation dye (Thermo) and co-cultured with macrophages at a 1:2 ratio for 4 days in RPMI supplemented with 20 ng/ml recombinant IL-2 (R&D Systems). Upon collection, samples were stained with LIVE/DEAD™ Fixable Near-IR Stain Dye (Thermo) and analyzed by flow cytometry. Proliferation modeling was performed using Flow-Jo 10.1 and results normalized to the T cell only control group.

Vural A., Nabar N.R., Hwang I.Y., Sohn S., Park C., Karlsson M.C., Blumer J.B, & Kehrl J.H. (2019). Gαi2 signaling regulates inflammasome priming and cytokine production by biasing macrophage phenotype determination. Journal of immunology (Baltimore, Md. : 1950), 202(5), 1510-1520.

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Multiparametric Flow Cytometry Analysis of Mediastinal Lymph Nodes

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Mediastinal lymph nodes (mLNs) were harvested, and single-cell suspensions were prepared. The cells were preincubated with Fc blocker and then incubated on ice for 30 min with antibody cocktail and Fixable Viability Dye, Ghost Red 780 (Tonbo Biosciences). Flow cytometry was conducted using fluorochrome-conjugated Abs for CD3 (145-2C-11, BD Biosciences), CD4 (RM5, BD Biosciences), CXCR5 (2G8, BD Biosciences), ICOS (C398.4A, BioLegend), PD-1 (J43, BD Biosciences and 29F.1A12, BioLegend), B220 (RA3–6B2, BD Biosciences), Fas (Jo2, BD Biosciences), PNA (FL-1071, Vector Laboratories), CD11b (M1/70, BD Biosciences), Ly6G (1A8, BioLegend), and rat IgG2a isotype control (BioLegend). Data were acquired using Fortessa (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). All data were analyzed with the FlowJo software (TreeStar, Ashland, OR).

Lama J.K., Iijima K., Kobayashi T, & Kita H. (2022). Blocking the inhibitory receptor PD-1 prevents allergic immune response and anaphylaxis in mice. The Journal of allergy and clinical immunology, 150(1), 178-191.e9.

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Mouse Spleen Cell Isolation Protocol

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Eight-week-old C57BL/6 mice were used for analysis. Cells from total spleen were dispersed by mechanical force. Resulting single-cell suspension was filtered through a 70-mm nylon mesh. Red blood cells were lysed using Ammonium-Chloride-Potassium lysing buffer (Lonza) at 1 ml per spleen for an incubation of 1 min on ice. In all subsequent steps, cells were incubated in 1× PBS containing 1% bovine serum albumin at 4°C and processed similar to the human cells as described above with the following exceptions. Fc block was anti-mouse (BD Biosciences). Primary antibody panel was as follows: CD3 (145–2C11, BD Biosciences), CD19 (1D3, BD Biosciences), CD11b (M1/70, BD Biosciences), CD21 (7G6, BD Biosciences), CD45R (RA3–6B2, Invitrogen), IgM (II/41, BD Biosciences), CD23 (B3B4, BioLegend), IgD (11026C.2a, BioLegend), and CD93 (AA4–1, BD Biosciences). A subset of experiments required the addition of CD39 (DUHA59) and CD73 (TY/118, BioLegend) to this panel.

Farmer J.R., Allard-Chamard H., Sun N., Ahmad M., Bertocchi A., Mahajan V.S., Aicher T., Arnold J., Benson M.D., Morningstar J., Barmettler S., Yuen G., Murphy S.J., Walter J.E., Ghebremichael M., Shalek A.K., Batista F., Gerszten R, & Pillai S. (2019). Induction of metabolic quiescence defines the transitional to follicular B cell switch. Science signaling, 12(604), eaaw5573.

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Tumor Immune Cell Analysis in MMTV-PyMT Mice

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The MMTV-PyMT model was prepared as described above. Mice were treated on day 7 with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg). Mice were euthanized on day 14. Cell suspensions were obtained from each tumor as described previously (9 (link)). Tumors were harvested and digested in DMEM supplemented with 2% FBS, collagenase D (2 mg/ml), and DNase I (40 μg/ml) (Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting the organs through a 70-μm cell strainer. Red blood cells were lysed with ACK lysing buffer (Quality Biological). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice before fixation. The following antibodies were used to stain the cells: CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8α (53-6.7, BD Biosciences), CD45 (30-F11, BD Biosciences), and NK1.1 (PK136, BD Biosciences). All flow cytometric analyses were done using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Sasaki K., Ishihara J., Ishihara A., Miura R., Mansurov A., Fukunaga K, & Hubbell J.A. (2019). Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy. Science Advances, 5(8), eaaw6081.

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Isolation and Stimulation of T-Cells

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Spleens were placed in ice cold, 4°C PBS and gently ground between frosted slides to produce a single-cell suspension. The suspension was centrifuged at 400×g for 10 min and the pellet was resuspended in PBS. Red blood cells were lysed with erythrocyte lysis buffer (BD Pharmingen, San Diego, CA) and the remaining cells were washed with PBS by centrifugation at 400×g for 10 min. Cell viability was consistently >95%, as determined using trypan blue exclusion procedure. CD4⁺ or CD8⁺ cells were purified by positive selection using CD4 or CD8 microbeads (>95% purity) obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). For cytokine production, purified CD4⁺ lymphocytes were cultured on plates coated with antibodies to 1 µg/ml of CD3 (145-2C11; BD Pharmingen) and 1 µg/ml of CD28 (37.51; BD Pharmingen) in RPMI 1640 medium (Invitrogen, Carlsbad, CA) with 10% heat-inactivated FBS (Invitrogen) at 37°C, 95% humidity, and 5% CO₂ for 24 h. After incubation, the cell-free suspension was collected and stored at −80°C until further analysis.

Lin C.W., Lo S., Hsu C., Hsieh C.H., Chang Y.F., Hou B.S., Kao Y.H., Lin C.C., Yu M.L., Yuan S.S, & Hsieh Y.C. (2014). T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis. PLoS ONE, 9(7), e102066.

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