Anti cd3 pe

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Anti-CD3-PE is a laboratory reagent used for the detection and analysis of CD3-positive cells. It consists of a phycoerythrin (PE) fluorescent dye conjugated to an antibody that specifically binds to the CD3 surface antigen expressed on T cells. This product can be used in flow cytometry applications to identify and quantify T cell populations within a sample.

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Close spelling variants of this product

Anti-CD3 (468 citations) CD3-PE (53 citations) PE anti-human CD3 (9 citations) PE-conjugated anti-CD3 (9 citations) Anti‐human CD3‐PE‐Cy7 (8 citations) Anti-CD3 PE/Dazzle 594 (6 citations) PE-Cy7-conjugated anti-CD3 (6 citations) PE/Cyanine7 anti-human CD3 (5 citations) PE anti-mouse CD3 antibody (5 citations) PE-Cy5-coupled anti-CD3 (4 citations)

39 protocols using anti cd3 pe

Tumor Infiltrating Immune Cell Analysis

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Tumor tissues, lymph nodes, and spleens were collected from mice after SDT and homogenized into single-cell suspensions following the established protocol (66 (link), 67 (link)). Then, the cell suspensions were stained with different antibodies according to the standard protocol. DCs in lymph nodes were stained with anti-CD11c–PE-Cy7 (BioLegend, catalog number 117318), anti-CD80–FITC (BioLegend, catalog number 104706), and anti-CD86–allophycocyanin (BioLegend, catalog number 105012) and analyzed by FCM (Thermo Fisher Scientific, Attune NxT).
To systematically investigate the in vivo antitumor immune responses against tumor metastasis, suspensions in tumors were stained with anti-CD3–PE (BioLegend, catalog number 100308), anti-CD8a–PerCP (BioLegend, catalog number 100732), and anti-CD4–allophycocyanin (BioLegend, catalog number 100412) antibodies. To further analyze T cells in spleens, cells were stained with anti-CD3–PE (BioLegend, catalog number 100308), anti-CD8a–PerCP (peridinin chlorophyll protein) (BioLegend, catalog number 100732), anti-CD4–allophycocyanin (BioLegend, catalog number 100412), anti-CD44–FITC (BioLegend, catalog number 103006), or anti-CD69–FITC (BioLegend, catalog number 104506). All these antibodies used in our experiments were in a 1:100 dilution from the stock solutions.

Wang L., Li G., Cao L., Dong Y., Wang Y., Wang S., Li Y., Guo X., Zhang Y., Sun F., Du X., Su J., Li Q., Peng X., Shao K, & Zhao W. (2021). An ultrasound-driven immune-boosting molecular machine for systemic tumor suppression. Science Advances, 7(43), eabj4796.

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Thymus T Cell Development Analysis

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To explore the development of T cells in thymus, single-cell suspensions in thymus were prepared and subsequently stained with the following anti-mouse antibodies: anti-CD4 (PE-CY7), anti-CD8 (BV510), anti-CD25 (APC) and anti-CD44 (AF700). The proportion of T and B cells in mice was determined by staining splenic cells with anti-mouse antibodies: anti-CD3 (PE) and anti-CD19 (PB450) antibodies (Biolegend). To detect the CD3⁺ T cell subsets, splenic cells were stained with the indicated anti-mouse antibodies: anti-CD3 (PE), anti-CD4 (PE-CY7), anti-CD8 (APC) (Biolegend). To detect T cell percentage in human peripheral blood, anti-human CD3 (FITC), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies were used. All these stained cells were evaluated using Beckman CytoFlex S system (Beckman).

Zhang Y., Du L., Wang C., Jiang Z., Duan Q., Li Y., Xie Z., He Z., Sun Y., Huang L., Lu L, & Wen C. (2024). Neddylation is a novel therapeutic target for lupus by regulating double negative T cell homeostasis. Signal Transduction and Targeted Therapy, 9, 18.

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Characterization of B-cell Differentiation

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LN biopsy samples were minced and evaluated by two-color flow cytometry after staining in phosphate-buffered-saline without calcium chloride magnesium chloride (PBS (−)) with the following monoclonal antibodies: anti-CD19-phycoerythrin (PE) (BioLegend, San Diego, CA, USA), anti-CD27-PE (BioLegend), anti-CD3-PE (BioLegend), anti-CD20-fluorescein isothiocyanate (FITC) (BioLegend), and anti-CD38-FITC (BioLegend). BiPSCs were also evaluated by two-color flow cytometry before and after the induction of hematopoietic differentiation using the following monoclonal antibodies: anti-CD19-PE, anti-CD27-PE, anti-CD34-PE (BioLegend), anti-CD20-FITC, anti-CD38-FITC, anti-CD43-FITC (BioLegend), and anti-CD45-FITC (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a BD FACSCANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Kawamura F., Inaki M., Katafuchi A., Abe Y., Tsuyama N., Kurosu Y., Yanagi A., Higuchi M., Muto S., Yamaura T., Suzuki H., Noji H., Suzuki S., Yoshida M.A., Sasatani M., Kamiya K., Onodera M, & Sakai A. (2017). Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells. Scientific Reports, 7, 1659.

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Thymus Cell Suspension Preparation and Staining

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Thymus cell suspensions were prepared and stained as described (Hager-Theodorides et al., 2005 (link)) using combinations of the following directly conjugated antibodies at concentration of 1:100: (from BD Pharmingen) anti-γδPE (catalogue no. 553178); from eBioscience: anti-TCRβFITC (catalogue no. 11-5961-85), antiCD3PE (catalogue no. 12-0031-82), anti-CD24PE (catalogue no. 12-0241-82) and anti-CD69FITC (catalogue no. 11-0691-85); (from Biolegend) anti-CD3FITC (catalogue no. 100204), anti-CD5FITC (catalogue no. 100605), anti-Qa2FITC (catalogue no. 121709), anti-CD4APC (catalogue no. 116014), anti-CD5PE (catalogue no. 100607), anti-CD8PerCP/Cy5.5 (catalogue no. 100734), anti-CD4PerCP/Cy5.5 (catalogue no. 100539) and anti-CD8APC (catalogue no. 100712). Data were acquired on a C6 Accuri flow cytometer (BD Biosciences) and analysed using FlowJo software. Live cells were gated by FSC and SSC profiles. Data represent at least three experiments.

Solanki A., Yanez D.C., Ross S., Lau C.I., Papaioannou E., Li J., Saldaña J.I, & Crompton T. (2018). Gli3 in fetal thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh. Development (Cambridge, England), 145(3), dev146910.

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Multiparameter Flow Cytometry Analysis

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These following antibodies (Abs) were used for surface marker staining and ICS combined with flow cytometry (all Abs were from Biolegend): anti-CD3-PErCP-cy5.5 (UCHT1), anti-CD3-FITC (UCHT1), anti-CD3-PE (HIT3a), anti-CD4-APC (OKT4), anti-CD4-APC-cy7 (RPA-T4), anti-CD8-PE (RPA-T8), anti-CD8-APC-cy7 (SK1), anti-CD14-FITC (HCD14), anti-IFN-γ-PE (4S.B3), anti-TNF-α-APC (MAb11), anti-IL-17A-PE-cy7 (BL168), anti-Foxp3-PE (206D), anti-TGF-β-PE-cy7 (TW4-2F8), anti-Perforin-PC (dG9), anti-granzyme A-PE (CB9),.

Tan Y., Guo W., Zhu Q., Song S., Xiang Y., Wu S., Zou S., Yan Y., Feng L., Luo M., Shen L., Feng Y, & Liang K. (2023). Characterization of peripheral cytokine-secreting cells responses in HIV/TB co-infection. Frontiers in Cellular and Infection Microbiology, 13, 1162420.

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Isolation and Characterization of Mouse Immune Cells

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The peripheral blood was obtained through the jugular vein of mice and mononuclear cells
were isolated through erythrocyte lysis. The cells were stained with anti-mCD45-FITC,
anti-CD45.2-BV711 (BD Bioscience, East Rutherford, NJ, USA), anti-CD3-PE, anti-CD3-APC,
anti-CD19-PE/Cy7, anti-CD11b-BV421 and anti-Gr-1-BV510 (BioLegend, San Diego, CA, USA) to
identify lymphoid and myeloid lineages of cells. Anti-CD132-PE (clone TUGm2, BioLegend)
was used to detect IL2RG protein. Flow cytometry was performed with BD LSR Fortessa II,
The BD FACS Aria Fusion (BD Bioscience).

Byambaa S., Uosaki H., Hara H., Nagao Y., Abe T., Shibata H., Nureki O., Ohmori T, & Hanazono Y. (2019). Generation of novel Il2rg-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9. Experimental Animals, 69(2), 189-198.

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Multiparameter Flow Cytometry of MAIT Cells

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100ul of whole blood was incubated for 2o minutes at room temperature in the dark with the following antibodies: anti-CD3 PE (317308, BioLegend, San Diego, USA), anti-CD161 PE-Cyanine 7(339918, BioLegend, San Diego, USA), anti-TCR Vα7.2 APC(351708, BioLegend, San Diego, USA), and anti-CD4 FITC(300505, BioLegend, San Diego, USA), incubated at room temperature, in the dark for 20 minutes. Cell lysis and fixation was performed with 1x RBC Lysis/Fixation Solution (422401, BioLegend, San Diego, USA) for 15 minutes at room temperature in the dark. After centrifuging at 350g for 5 minutes, supernatant was discarded, then washed twice with Cell Staining Buffer. The cell pellet was resuspended in 500µl Cell Staining Buffer (420201, BioLegend, San Diego, USA) and analyzed on BD FACSCalibur flow cytometer using the CellQuestPro Software (BD Biosciences). Data were analyzed using FlowJo Version 10.5.3 software (TreeStar). The serum obtained after centrifugation was stored at -80°C.

Zhou H., Xu J., Hong L., Jia Y., Burk L.V., Chi F., Zhao M., Guan X., Liu D., Yin X., Zhang Y., Teng X., Duan L, & Li K. (2022). The alterations of circulating mucosal-associated invariant T cells in polycystic ovary syndrome. Frontiers in Endocrinology, 13, 1038184.

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Detailed Co-Incubation Assay for Immune Cell Interactions

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For experiments using F5-Jurkat, 1G4-Jurkat, MART1-K562 and NYESO-K562 cells, co-incubations were set up in 96-well U-bottom plates at a ratio of 5:1 Jurkat to K562 (300,000 cells per well) and co-incubated for 45 min at 37 °C. Ratios for co-incubation experiments involving SCT-Jurkat cells and TCR-K562 cells are indicated in the text and figure legends. The co-incubation was then centrifuged for 5 min at 1,500 r.p.m. and the medium was aspirated by vacuum. The cell pellets were resuspended with cold PBS solution containing 2 mM EDTA and then centrifuged. Cells were stained with different antibodies at 4 °C for 20 min, washed twice, and then analyzed by flow cytometry using MACSQuant Analyzer 10 (Miltenyi Biotec). The following antibodies were used to detect expression of surface markers: anti-CD3-PE, anti-CD8-BV510, anti-TCRαβ-PacificBlue, anti-TCRαβ-PE/Cy7, anti-LNGFR-PE, anti-LNGFR-APC, anti-HLA-A2-PacificBlue, anti-HLA-A2-BV510, and anti-muTCRa β-PE/Cy7 (all from Biolegend). Flow cytometry gating strategies are described in the Supplementary Note.

Li G., Bethune M.T., Wong S., Joglekar A.V., Leonard M.T., Wang J.K., Kim J.T., Cheng D., Peng S., Zaretsky J.M., Su Y., Luo Y., Heath J.R., Ribas A., Witte O.N, & Baltimore D. (2019). T cell antigen discovery via trogocytosis. Nature methods, 16(2), 183-190.

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Multiparametric Cell Sorting Protocol

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All cell populations were sorted using a FACSAria™ III (BD Biosciences, Franklin Lakes, NJ), and only preparations with a purity of >95% were used for further experiments. Memory CD4⁺ and CD8⁺ T cells were sorted from peripheral blood after staining with the following antibodies: anti-CD3-PE (Biolegend, San Diego, CA), anti-CD4-APC (eBiosciences, San Diego, CA), anti-CD8-Pacific Blue (Biolegend) and anti-CD45RO FITC (Biolegend). T cells expressing specific Vβ families were sorted from bulk CSF Phytohaemagglutinin (PHA)-expanded cells after staining with the corresponding TCR Vβ-specific antibodies22 (link) (Beckman Coulter, Nyon, Switzerland). B cells from peripheral blood were sorted after staining with anti-CD19-Pacific Blue (BD).

Planas R., Metz I., Ortiz Y., Vilarrasa N., Jelčić I., Salinas-Riester G., Heesen C., Brück W., Martin R, & Sospedra M. (2015). Central role of Th2/Tc2 lymphocytes in pattern II multiple sclerosis lesions. Annals of Clinical and Translational Neurology, 2(9), 875-893.

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Immune Cell Profiling in Tumor-Bearing Mice

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TdLNs and tumors from mice with different treatments were collected for flow cytometry analysis. Briefly, single‐cell suspensions were prepared by digestion with collagenase type IV (2.5 mg mL⁻¹, Gibco, USA) in Hank's Balanced Salt Mixture containing calcium and magnesium (Solarbio, China) for 1.5 h at 37 °C. Next, the cells were then diluted to a concentration of 1 × 10⁷ cells/mL in a Cell Staining Buffer (BioLegend, USA). Cell samples were then stained with anti‐CD3‐PE, anti‐CD4‐APC‐Cy7, anti‐CD8‐APC, anti‐CD11c‐APC, anti‐CD86‐PerCP, and anti‐103‐PE for 1 h in the dark (BioLegend, USA). Then, the cells were washed twice with Cell Staining Buffer, and analyzed with Beckman CytoFLEX. Flow cytometry data were analyzed using CytExpert software. Percentages of CTLs (CD3⁺CD4^‐CD8⁺), helper T cells (CD3⁺CD4⁺CD8^‐), and mature dendritic cells (CD11c⁺CD103⁺CD86⁺) were calculated.

Ji P., Sun W., Zhang S., Xing Y., Wang C., Wei M., Li Q., Ji G, & Yang G. (2023). Modular Hydrogel Vaccine for Programmable and Coordinate Elicitation of Cancer Immunotherapy. Advanced Science, 10(22), 2301789.

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