Cd3 145 2c11

Manufactured by BioLegend

Sourced in United States

CD3 (145-2C11) is a monoclonal antibody that recognizes the CD3 complex on T cells. The CD3 complex is a key component of the T cell receptor (TCR) and is essential for T cell activation and signaling.

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Lab products found in correlation

Cd11b m1 70, by BioLegend (6 mentions) Cd45 30 f11, by BioLegend (5 mentions) Cd8 53 6, by BioLegend (4 mentions) Cd4 rm4 5, by BioLegend (4 mentions) Cd19 6d5, by BioLegend (4 mentions) Cd86 gl 1, by BioLegend (4 mentions) Cd69 h1.2f3, by BioLegend (4 mentions) Lsrfortessa, by BD (4 mentions) Foxp3 fjk 16s, by Thermo Fisher Scientific (4 mentions) Cd4 gk1, by BioLegend (3 mentions) Cd11c n418, by BioLegend (3 mentions) Ly6g 1a8, by BioLegend (3 mentions) Ly6c hk1, by BioLegend (3 mentions) Cd25 pc61, by BioLegend (3 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (3 mentions) Ly6c hk1, by Thermo Fisher Scientific (2 mentions) Cd45.2 104, by BioLegend (2 mentions) Live dead fixable blue, by Thermo Fisher Scientific (2 mentions) Cytofix cytoperm buffer set, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions)

Close spelling variants of this product

Anti-CD3 (clone 145-2C11, (34 citations) Anti-CD3 (145-2C11; (27 citations) CD3 (clone 145-2C11, (9 citations) Anti-CD3 antibody (145-2C11; (6 citations) CD3-PerCP/Cy5.5 (145-2C11; (5 citations) Anti-CD3 mAb (clone 145-2C11, (5 citations) CD3-FITC clone 145–2C11 (5 citations) Anti-CD3 antibody (clone 145-2C11, (3 citations) Anti-mouse CD3 (145-2C11; (3 citations) Anti-CD3-PerCP (clone 145-2C11, (3 citations)

24 protocols using cd3 145 2c11

T Cell Phenotyping by Flow Cytometry

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To confirm the outcome of the T cell transfer studies spleen cells were stained with monoclonal antibodies against the following molecules: CD3 (145-2C11), CD4 (GK1.5) and CD8 (53–6.7) from Biolegend (San Diego, CA). Surface stained cells were measured by an Accuri C6 flow cytometer (BD Biosciences) or a FACS LSRII (Becton Dickinson, San Jose, CA), and analyzed using C6 software or FlowJo software (TreeStar, Ashland, OR).

Robinson J.W., Li J.Y., Walker L.D., Tiyagi A.M., Reott M., Yu M., Adams J., Weitzmann M.N, & Pacifici R. (2015). T CELL EXPRESSED CD40L POTENTIATES THE BONE ANABOLIC ACTIVITY OF INTERMITTENT PTH TREATMENT. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 30(4), 695-705.

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Dissection and Analysis of Murine Pancreatic Immune Cells

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Prior to organ dissection, mice were perfused with 20 ml PBS to eliminate contaminating blood leukocytes. Single-cell suspensions of the pancreata were prepared by Collagenase P (Roche) digestion. Cells from pancreatic lymph nodes and spleen were prepared by physical dissociation. The spleen was treated with ACK lysing buffer (Thermo Fisher Scientific). All stainings began with an incubation with TruStain fcX anti-mouse CD16/32. Antibodies used for subsequent stainings were: anti-CD45 (30-F11), -CD19 (6D5); -CD3 (145–2C11), -CD4 (RM4–5), -CD8 (53–6.7), -CD25 (PC61), CD11b (M1/70), -CD11c (N418), -F4/80 (BM8), and -Gr1 (RB6–8C5) (all from BioLegend). Intracellular Foxp3 (FJK-16s) staining was performed according to eBioscience’s protocol. Samples were acquired with an Attune NxT flow cytometer (Thermo Fisher Scientific) and data were analyzed with FlowJo software (Tree Star, Inc.).

Lee H., Lee Y.S., Harenda Q., Pietrzak S., Oktay H.Z., Schreiber S., Liao Y., Sonthalia S., Ciecko A.E., Chen Y.G., Keles S., Sridharan R, & Engin F. (2020). Beta cell dedifferentiation induced by IRE1α deletion prevents type 1 diabetes. Cell metabolism, 31(4), 822-836.e5.

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Histological and Flow Cytometry Analysis of Kidney

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For IHC, kidneys were fixed with 10% buffered formaldehyde and embedded in paraffin. Slices of 4 μm thick were stained with H&E or periodic acid–Schiff (PAS) and observed on an EVOS microscope (Thermo Fisher Scientific). The slides were scored blindly by an expert with more than 10 years of experience with renal histopathology, as described previously (62 (link)). Briefly, severity of GN was assessed by mild to moderate increase in mesangial cellularity, thickening of the GBM, endocapillary hypercellularity, and crescents formation. The tubulointerstitial inflammation was measured by assessing tubular atrophy and tubulointerstitial inflammation.
Kidneys were perfused with PBS containing EDTA (MilliporeSigma) before harvesting. Kidneys were digested at 37°C in 1 mg/mL collagenase IV (Worthington) in complete RPMI for 30 minutes, filtered through 70 mm strainers, and washed twice in PBS. For flow cytometry, the following antibodies were used: CD45 (30-F11, eBioscience), CD3 (145-2C11, BioLegend), CD4 (GK1.5, BioLegend), CD8 (53-6.7, BioLegend), CD45R/B220 (RA3-6B2, BioLegend), Ly6G (IA8, eBioscience), CD11b (M1/70, BioLegend), F4/80 (BM8, BioLegend), and Ly6C (HK1.4, eBioscience). Samples were acquired on BD LSRFortessa cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc.).

Li D.D., Bechara R., Ramani K., Jawale C.V., Li Y., Kolls J.K., Gaffen S.L, & Biswas P.S. (2021). RTEC-intrinsic IL-17–driven inflammatory circuit amplifies antibody-induced glomerulonephritis and is constrained by Regnase-1. JCI Insight, 6(13), e147505.

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Multicolor Flow Cytometry Panel

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Antibodies were conjugated to fluorochromes (FITC, PE, PECy7, APC, APCCy7, Pacific Blue, and BV711) and were specific for the following mouse antigens: CD3 (145-2C11; Biolegend), CD11b (M1/70; Sony), CD11c (HL3; Biolegend), CD19 (6D5; Sony), CD45.2 (104; Biolegend), Ly6C (Hk1.4; eBioscience), Ly6G (RB6-8C5; BD Pharmingen), CD45 (30F11; Sony), and TER119 (Ter119; BD Pharmingen).

Cardoso A., Gil Castro A., Martins A.C., Carriche G.M., Murigneux V., Castro I., Cumano A., Vieira P, & Saraiva M. (2018). The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis. Frontiers in Immunology, 9, 400.

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Murine Immune Cell Phenotyping

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Murine-specific antibodies were purchased from BD Biosciences: (CD3: 145-2C11; PD-L1/CD247: MIH5; TIM-3: RMT3-23; PD-1: J43; LAG-3: C9B7W) and from BioLegend: (CD-45: 30F-11, CD3: 2C11, B220: RA3-6B2, CD11b: M1/70, CD11c: HL3, NK1.1 PK136). Appropriate isotype controls were used when applicable. The LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude dead cells. Cells were analyzed with an LSRFortessa (BD Biosciences) and data were analyzed with FlowJo Software (TreeStar).

Kemeny H.R., Elsamadicy A.A., Farber S.H., Champion C.D., Lorrey S.J., Chongsathidkiet P., Woroniecka K.I., Cui X., Shen S.H., Rhodin K.E., Tsvankin V., Everitt J., Sanchez-Perez L., Healy P., McLendon R.E., Codd P.J., Dunn I.F, & Fecci P.E. (2019). Targeting PD-L1 Initiates Effective Antitumor Immunity in a Murine Model of Cushing Disease. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(5), 1141-1151.

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Monoclonal Antibodies for Immune Cell Analysis

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The following monoclonal antibodies were purchased from eBiosciences, BD Pharmingen or BioLegend: CD3 (145-2C11), CD4 (RM4-5), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CXCR5 (L138D7), NPR-1 (3E12), ST2 (RMST2-2), TCRβ (H57-597), TCR Vβ6 (RR4-7), TCR Vβ8.1/8.2 (MR5-2), TCR Vβ14 (14-2), Bcl-6 (K112-91), c-Maf (T54-853), Foxp3 (FJK-16s), GATA3 (TWAJ), Helios (22F6), RORγt (B2D or Q31-378), T-bet (eBio4B10), IL-10 (JES5-16E3), IL-17A (eBio17B7) and IFN-γ (XM61.2). 4′,6-diamidino-2-phenylindole (DAPI) or Live/dead fixable blue (ThermoFisher) was used to exclude dead cells.
For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine analysis, cells were incubated for 5 h in RPMI with 10% FBS, phorbol 12-myristate 13-acetate (PMA) (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma) and GolgiStop (BD). Cells were stained for surface markers before fixation and permeabilization, and then subjected to intracellular cytokine staining according to the manufacturer’s protocol (Cytofix/Cytoperm buffer set from BD Biosciences).
Flow cytometric analysis was performed on an LSR II (BD Biosciences) or an Aria II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Xu M., Pokrovskii M., Ding Y., Yi R., Au C., Harrison O.J., Galan C., Belkaid Y., Bonneau R, & Littman D.R. (2018). c-Maf-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont. Nature, 554(7692), 373-377.

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Murine Lymphoid Tissue Dissociation

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Cell suspensions were obtained from the spleen, the PP, the MLN and the AA LN (lumbar and renal lymph nodes). Jejunum and colon sections from PBS and LCWE-injected mice were harvested and dissociated into single-cell suspensions with a gentleMACS™ Octo Dissociator (Miltenyi Biotec) and a mouse Lamina Propria Dissociation kit (Miltenyi Biotec) following the complete protocol from the manufacturer. The following antibodies against the respective murine antigens were used: IgA (mA-6E1,Thermo Fisher Scientific), CD19 (eBio1D3, Thermo Fisher Scientific), CD4 (RM4–5, Tonbo Biosciences), CD3 (145–2C11, BioLegend and Tonbo Biosciences), CD45.1 (A20, Thermo Fisher Scientific), CD45.2 (104, BioLegend), CD95 (SA367H8, BioLegend), GL-7 (GL-7, BioLegend). Dead cells were routinely excluded based on the staining of Fixable Viability dye (FVD) eFluor 506 (Thermo Fisher Scientific). Cell numbers were calculated by flow cytometry with the CountBright Absolute Counting Beads (Thermo Fisher Scientific). Stained cells were analyzed on a LSRII (BD Biosciences) and the data were processed using Flowjo (Tree Star Inc.).

Rivas M.N., Wakita D., Franklin M.K., Carvalho T.T., Abolhesn A., Gomez A.C., Chen S., Lehman T.J., Sato K., Shibuya A., Fasano A., Kiyono H., Abe M., Tatsumoto N., Yamashita M., Crother T.R., Shimada K, & Arditi M. (2019). Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation. Immunity, 51(3), 508-521.e6.

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CD8+ T Cell Activation Assay

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3 × 10⁶ CD8⁺ T cells were incubated with biotinylated antibodies against CD3 (145-2C11, 1 μg/ml), anti-CD8 (53-6.7, 10 μg/ml) and anti-CD28 (37.51, 2 μg/ml) (all from Biolegend) on ice for 30 min in 85 μl RPMI with 0.5% FCS. Cells were warmed at 37°C for two minutes before adding 85 μl 1/50 streptavidin (Jackson Immunoresearch #016-000-084) and incubating for 5 minutes at 37°C. The cells were pelleted by centrifugation and resuspended in 50 μl ice cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM NaF, 10 mM NaF, 10 mM Indoacetamie, 5 μl Protease inhibitor (PROTEOLOC), 5 μl of EDTA (PROTEOLOC), 1% (w/v) NP40(BDH)) and incubated for 10 min on ice.
Lysates were centrifuged at 14,800 rpm at 4°C in a microcentrifuge. The supernatant was subsequently mixed with 16.7 μl NuPAGE LDS sample buffer (Life Technologies) and frozen. Lysates were thawed at room temperature and 1 μl of 1M DTT (Life Technologies) was added. The proteins were separated on a 10% Bis-Tris gel (Life Technologies) and transferred to PVDF membranes which were probed with the indicated antibodies, all from eBioscience except anti-β-actin which was from SantaCruz.

Pearce V.Q., Bouabe H., MacQueen A.R., Carbonaro V, & Okkenhaug K. (2015). PI3Kδ Regulates the Magnitude of CD8+ T Cell Responses after Challenge with Listeria monocytogenes. The Journal of Immunology Author Choice, 195(7), 3206-3217.

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Flow Cytometry Immune Cell Profiling

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Single-cell suspensions were stained
with the fixable viability dye eFluor 780 (eBioscience) for 30 min
at 4 °C, and Fc receptors were blocked with an anti-CD16/32 (BioLegend)
blocking antibody prior to surface staining with monoclonal antibodies.
Antibodies used for surface staining are as listed: mouse: CD45 (30-F11,
BioLegend), lineage (Lin) markers [CD3 (145-2C11, BioLegend), CD19
(6D5, BioLegend), CD11b (M1/70, BioLegend), CD11c (N418, BioLegend)],
and CD86 (GL-1, BioLegend). For CFSE staining, cells were stained
with 5 μM CFSE according to the manufacturer’s protocol.
Data were acquired on LSR II (BD Biosciences) and analyzed with FlowJo
v. 10.1 software (TreeStar).

González-Cuesta M., Lai A.C., Chi P.Y., Hsu I.L., Liu N.T., Wu K.C., García Fernández J.M., Chang Y.J, & Ortiz Mellet C. (2023). Serine-/Cysteine-Based sp2-Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma. Journal of Medicinal Chemistry, 66(7), 4768-4783.

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Multiparameter Flow Cytometry of Immune Cells

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Flow cytometric analysis was performed on a Canto II flow cytometer (BD Biosciences) using FlowJo software v10.7.1 (Tree Star Inc., Ashland, OR, USA). Dead cells were excluded using a live/dead fixable dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA). The following mouse-specific antibodies were used for staining: CD3 (145-2C11, BioLegend, San Diego, CA, USA), CD4 (RM4-5, BioLegend), Foxp3 (FJK-16s, Invitrogen), T-bet (4B10, Invitrogen, Eugene, OR, USA), RORγt (B2D, Invitrogen), GATA3 (16E10A23, BioLegend), Siglec F (S17007L, Biolegend), Ly6G (1A8, Biolegend), CD11b (M1/70, BioLegend), B220 (RA3-6B2, BioLegend), IgA (C10-3, Biolegend), and IgM (RMM-1, Biolegend).

Gu B.H., Rim C.Y., Lee S., Kim T.Y., Joo S.S., Lee S.J., Park H.K, & Kim M. (2022). Alteration of Gut Immunity and Microbiome in Mixed Granulocytic Asthma. Biomedicines, 10(11), 2946.

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