Anti cd3

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-CD3 is a monoclonal antibody that binds specifically to the CD3 complex on the surface of T cells. The CD3 complex is a key component of the T cell receptor (TCR) and plays a crucial role in T cell activation and signaling.

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10 protocols using anti cd3

Examining PSGL-1 Signaling in Activated CD8+ T Cells

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At 9-dpi, 2 × 10⁶ splenocytes from WT mice were cultured for 96hr in complete medium supplemented 2-ME and 50 U/mL IL-2 under the following conditions: 10μg/mL rat IgG (Jackson ImmunoResearch), 10μg/mL anti-PSGL-1 (4RA10, BioXCell), 2μg/mL GP_33-41 peptide or both anti-PSGL-1 and peptide. Cultured cells were tetramer stained and live cells determined by excluding PI⁺ cells. For pSTAT-5 staining, cells were incubated with 5μg/mL of IgG or anti-PSGL-1 and stimulated with GP_33-41 peptide as above. Additionally, responding CD8⁺ T cells (CD8⁺CD44⁺CD11a⁺CD49d⁺) were FACS-sorted from spleens of CL13 infected mice at 9-dpi to >96% purity. The cells were stimulated for the indicated times with plate bound anti-CD3 (10μg/mL) and plate bound rat IgG or anti-PSGL-1 followed by immunoblotting with antibodies specific for phospho ERK1/2-Thr202/Tyr204, phospho AKT-Ser473, ERK1/2, and AKT (all from Cell Signaling Technologies).

Tinoco R., Carrette F., Barraza M.L., Otero D.C., Magaña J., Bosenberg M.W., Swain S.L, & Bradley L.M. (2016). PSGL-1 is an immune checkpoint regulator that promotes T cell exhaustion. Immunity, 44(5), 1190-1203.

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Allogeneic Kidney Graft Histology

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Kidney grafts were harvested at designated time points and either immediately submerged in 10% formalin for paraffin embedding or preserved in OCT; and 4 μm sections were used for HE staining. IHC and immunofluorescence (IF) staining were performed as published previously (38 (link)). The Abs used included anti-CD3(Cell Signaling Technology, D4V8L), B220 (BioLegend, RA3-6B2), and C4d (Novus Biologicals, 16D2). To test the ability of DSA produced from single-cell cultures to bind to alloantigens, supernatants from the highest 5 DSA-positive clones were pooled and used to bind allogeneic native Balb/c kidney sections for 24 hours at 4°C. Then, primary Ab anti-IgG (SouthernBiotech) was used to detect the IgG. Images were captured on a ZEISS Axiolab 5 microscope.

Zhang H., Cavazzoni C.B., Podestà M.A., Bechu E.D., Ralli G., Chandrakar P., Lee J.M., Sayin I., Tullius S.G., Abdi R., Chong A.S., Blazar B.R, & Sage P.T. (2023). IL-21–producing effector Tfh cells promote B cell alloimmunity in lymph nodes and kidney allografts. JCI Insight, 8(20), e169793.

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Histological Assessment of Skin Changes

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For histologic evaluation of epidermal thickness, LI, and the number of dermal infiltrated inflammatory cells, groups of mice (n = 4 mice/group) were treated with the triterpene compounds according to the short-term protocol regimen. BrdU (100 μg/g B.W.) dissolved in PBS was injected i.p. to mice 30 min prior to sacrifice. Forty-eight hrs after the last TPA treatment, dorsal skin was excised and fixed in 10% neutral-buffered formalin. The fixed skin samples were embedded in paraffin, sectioned (4 μm) and stained with toluidine blue O (Fisher Scientific, Pittsburgh, PA), anti-BrdU (Abcam, Cambridge, MA), or anti-CD3 (Cell Signaling Technology, Beverly, MA). Epidermal thickness and LI were determined as described previously [62 (link)]. The number of inflammatory cells in the dermis was counted per 200 mm² field as previously described [59 (link)].

Cho J., Tremmel L., Rho O., Camelio A.M., Siegel D., Slaga T.J, & DiGiovanni J. (2015). Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Oncotarget, 6(36), 39292-39306.

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Histological and Immunohistochemical Analysis of Colon Tissue

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In brief, fresh colon tissues were fixed in 4% paraformaldehyde at 4 °C overnight, then subjected to paraffin imbedding sections. For H&E Staining, the sections were stained with haematoxylin and dehydration in graded alcohols and xylene. For immunolabeling, the sections were incubated with indicated primary antibodies: anti-CD3 [1:400, 85061; Cell Signaling]; anti-Ki67 [1:200, GB13030-2; Servicebio], anti-F4/80 [1:200, GB11027; Servicebio], anti-ChgA [1:400, ab45179; Cell Signaling] anti-CAI [1:200, SC39349; Santa Cruz], anti-Lysozyme [1:300, ab108502; Abcam], anti-N2ICD [1:200, YC0069; Immunoway], overnight at 4 °C in the dark. Then, the sections were incubated with either HRP–conjugated Goat anti-Rabbit IgG (1:200, G1215; Servicebio) or HRP–conjugated Goat anti-Mouse IgG (1:200, G1214; Servicebio) for 50 min at 25 °C. The subsequent detection was performed using the standard substrate detection of DAB. TUNEL assay kit was purchased from Abcam (ab66110). Images were taken by using Leica DM6 B Upright Microscope.

Pu Y., Song Y., Zhang M., Long C., Li J., Wang Y., Xu Y., Pan F., Zhao N., Zhang X., Xu Y., Cui J., Wang H., Li Y., Zhao Y., Jin D, & Zhang H. (2021). GOLM1 restricts colitis and colon tumorigenesis by ensuring Notch signaling equilibrium in intestinal homeostasis. Signal Transduction and Targeted Therapy, 6, 148.

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Allogeneic Kidney Graft Histology

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Comprehensive Hematological and Immunological Profiling

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CBC was measured by an automated blood count (Hemavet System 950FS). May-Grünwald-Giemsa staining (Thermo Fisher Scientific, catalog 22-050-272) was used to stain PB smears. Histological analyses were performed as previously described (83 (link)), and samples were stained with H&E.
The following antibodies were used for IHC analysis: anti-CD68 (Leica Biosystems, PA0273); anti-CD69 (Abcam, 202909); anti-Ki67 (Cell Signaling Technology, 12202S; Leica Biosystems, PA0230) anti-galectin/MAC2 (Abcam, ab76245); anti-CD3 (Cell Signaling Technology, 99940S); anti-CD4 (Leica Biosystems, PA0427); anti-CD8 (Leica Biosystems, PA0183); and anti-FOXP3 (Abcam, ab215206). For BM and liver fibrosis analyses, a reticulin staining kit (MilliporeSigma, HT102A-1KT) was used.
For plasma analyses, PB samples were collected in heparinized tubes and centrifuged, and plasma was frozen or directly assayed. Plasma was used for the cytokine array analyses (R&D Systems, ARY028); the ferritin ELISA kit (ALPCO, 41-FERMS-E01); the sCD25 ELISA kit (G-Biosciences, IT5809); and chemistry profiling (HESKA; catalog 6330, COMP/EWRAP).

Mas G., Man N., Nakata Y., Martinez-Caja C., Karl D., Beckedorff F., Tamiro F., Chen C., Duffort S., Itonaga H., Mookhtiar A.K., Kunkalla K., Valencia A.M., Collings C.K., Kadoch C., Vega F., Kogan S.C., Morey L., Bilbao D, & Nimer S.D. (2023). The SWI/SNF chromatin-remodeling subunit DPF2 facilitates NRF2-dependent antiinflammatory and antioxidant gene expression. The Journal of Clinical Investigation, 133(13), e158419.

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Immunofluorescence Analysis of Skeletal Muscle

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Skeletal muscle sections were de-paraffinized in xylene, rehydrated in graded ethanol and phosphate-buffered saline solution, and antigen-unmasked with sodium citrate (10 mmol/L, pH = 6.0) followed by phosphate-buffered saline (PBS) wash and blocking with 2%bovine serum albumin in phosphate-buffered saline at room temperature for 2 hours.^{17 (link),18 (link)} After phosphate-buffered saline wash, overnight incubation with anti- CD3 (Cell Signaling), VE-cadherin (Cell Signaling), and anti-phospho-VE-cadherin (Lifespan Biosciences Inc. Seattle, WA) antibodies (each used 1:50) was performed at 4°C. Anti-mouse, smooth muscle actin (1:1000; Sigma, St Louis, Mo) was used to detect microvascular smooth muscle. Sections were then washed in phosphate-buffered saline and incubated with the appropriate Alexa fluor secondary antibody and mounted using fluorescent mounting medium (Vector Labs, Burlingame, Calif). Tissue labeling with secondary antibody alone or with normal rabbit IgG or serum in place of primary antibody served as a negative control. Tissue was visualized using a Nikon E800 epi-fluorescent microscope system (Nikon Inc. Melville, NY). Six image photos per slide were taken at same magnificent resolution (20 x Plan Fluor objective) for optical density (intensity) analysis (Spot RT3, Diagnostic Instruments, Sterling Heights MI)

Feng J., Cohn W.E., Parnis S.M., Sodha N.R., Clements R.T., Sellke N., Frazier O.H, & Sellke F.W. (2015). New Continuous-Flow Total Artificial Heart and Vascular Permeability. The Journal of surgical research, 199(2), 296-305.

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Immunofluorescence Analysis of T-Cell Markers

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Allograft tissues from the syngeneic mice were collected and fixed in 10% (v/v) formalin overnight and embedded in paraffin. Formalin-fixed paraffin embedded (FFPE) tissue samples were sectioned onto microscope slides, which were dewaxed and rehydrated sequentially in a graded ethanol series (100%, 90%, 70% and 50%) and finally in 1×PBS for 3 minutes per wash. Antigen retrieval was performed using antigen unmasking solution (Vector Laboratories, #H-3300). Slides were incubated with anti-CD3 (Cell Signaling, #78588) or anti-CD8 (Cell Signaling, #98941) antibody overnight at 4°C, followed by Alexa Fluor 488 Goat anti-Rabbit IgG secondary antibody (Invitrogen, #A11034) for 1 hour at RT. After washing with 1×PBS for three times, tissues were mounted by ProLong Gold Antifade Mountant with DAPI (Invitrogen, #P36931). Images were captured on a confocal laser scanning microscope (Carl Zeiss, LSM710).

Hong J., Lee J.H., Zhang Z., Wu Y., Yang M., Liao Y., de la Rosa R., Scheirer J., Pechacek D., Zhang N., Xu Z., Curiel T., Tan X., Huang T.H, & Xu K. (2022). PRC2-mediated epigenetic suppression of type I IFN-STAT2 signaling impairs antitumor immunity in luminal breast cancer. Cancer research, 82(24), 4624-4640.

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Immunohistochemical Analysis of Pancreatic Tissues

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Paraffin-embedded pancreas tissue sections were dewaxed and rehydrated twice in PBS for 15 min. The sections were incubated with 0.3% H₂O₂ for 15 min to block endogenous peroxidases, washed twice in PBS, then incubated with 30 g/L of BSA for 10 min to prevent non-specific binding of antibodies, and then incubated with anti-CD3 (catalog # 78588, Cell Signaling Technology) or anti-F4/80 (catalog # 25514, Cell Signaling Technology). Finally, diaminobenzidine (DAB) was added as chromogen followed by hematoxylin. The images were observed by a light microscope, and the positive areas were analyzed by ImageJ software.

Zhang D., Li W., Wang M., Yin H., Xia C., Li K, & Huang H. (2022). Methods of a New Chronic Pancreatitis and Spontaneous Pancreatic Cancer Mouse Model Using Retrograde Pancreatic Duct Injection of Dibutyltin Dichloride. Frontiers in Oncology, 12, 947133.

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Immunofluorescence Staining of CD3+ Cells

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After dewaxing and hydrating the paraffin-embedded slices (5 μm), the antigen was recovered with sodium citrate (Beyotime Biotechnology, Nanjing, China). After blocking the sections with 5% goat serum (Proteintech, Rosemount, PA, USA), tissue slices were treated with the anti-CD3 (1:100, Cell Signaling Technology, Danvers, MA, USA) antibody at 4 °C overnight. Before immunofluorescence dye, secondary antibodies: goat anti-rabbit IgG2a coupled to Alexa Fluor 488 (1:200, Invitrogen, Waltham, MA, USA) were applied to probe the slices. The sections were photographed using a fluorescence microscope (Olympus, Tokyo, Japan) after being counterstained with DAPI (Abcam, Cambridge, UK).

Han Y., Yang H., Hua Z., Nie S., Xu S., Zhou C., Chen F., Li M., Yu Q., Sun Y., Wei Y, & Wang X. (2023). Rotating Magnetic Field Mitigates Ankylosing Spondylitis Targeting Osteocytes and Chondrocytes via Ameliorating Immune Dysfunctions. Cells, 12(7), 972.

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