Pe cy7 anti mouse cd3

Manufactured by BioLegend

Sourced in United States

PE-Cy7-anti-Mouse CD3 is a flow cytometry reagent that binds to the CD3 receptor on mouse T cells. It is conjugated with the fluorescent dye PE-Cy7 for detection by flow cytometry.

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Close spelling variants of this product

Anti-CD3 (468 citations) CD3-PE-Cy7 (67 citations) CD3-PE (53 citations) Anti-CD3-PE-Cy7 (52 citations) Anti-mouse CD3 (46 citations) Anti-CD3-PE (39 citations) PE anti-mouse CD3 (16 citations) PE/Cy7)-conjugated anti-mouse CD3 (3 citations)

9 protocols using pe cy7 anti mouse cd3

Tumor Immune Profiling in Mice

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Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Liu Z.Y., Zhang D.Y., Lin X.H., Sun J.L., Abuduwaili W., Zhang G.C., Xu R.C., Wang F., Yu X.N., Shi X., Deng B., Dong L., Weng S.Q., Zhu J.M., Shen X.Z, & Liu T.T. (2022). Nalidixic acid potentiates the antitumor activity in sorafenib-resistant hepatocellular carcinoma via the tumor immune microenvironment analysis. Frontiers in Pharmacology, 13, 952482.

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Comprehensive T-cell Phenotyping by Flow Cytometry

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Single-cell suspension (10⁶ cells/tube) was incubated with anti-CD16/32 antibody to block Fc receptors for 15min at 4°C. For surface staining, the cells were stained with antibodies or matched isotype control for 20 min at 4°C in the dark. The antibodies included anti-mouse CD3-PECY7, CD4-FITC, CD8-Percpcy5.5, CD25-APC, CD11c-PE, CD86-APC, CD80-PerCP-eFlour710, PD-L1-PECY7, MHCII-Alexa Flour 700, MHCII- APC, and CD103-FITC antibodies (all from Biolegend, San Diego, CA, USA). For intracellular staining, cells were cultured in the presence of 2μl/ml cell stimulation cocktail (eBioscience) for 6 h. The cells were fixed and permeabilized using fixation and permeabilization solutions, then stained with anti-mouse Foxp3-PE, IL4-PE, IFN-γ-APC, IL10-PE antibodies (all from eBioscience), or isotype control for 30 min at 4°C in the dark. The cells were detected using BD FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA) or Aurora (Cytek, Fremont, CA, USA), and data were analyzed using the FlowJo 10.0.7 software (Tree Star, Ashland, OR, USA).

Xu B., Ling S., Xu X., Liu X., Wang A., Zhou Y., Luo Y., Li W, & Yao X. (2021). A New Formulation of Probiotics Attenuates Calcipotriol-Induced Dermatitis by Inducing Regulatory Dendritic Cells. Frontiers in Immunology, 12, 775018.

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Characterization of Mouse T Cells

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Mice were administrated with or without complex 1 (5.0 mg kg⁻¹) for 6 h, anesthetized, and T cells were extracted from the spleen as previously described [82 (link)]. T cells were incubated with antibodies for surface markers, including anti-mouse-CD3-PE-Cy7, anti-mouse-CD4-FITC, and anti-mouse-CD8-PE antibodies (BioLegend, Shanghai, China) for 20 min, then the cells were accounted 10⁴ cells and determined using flow cytometry analysis. The acquisition was performed with FACSpCanto II using the FACSDiva software (BD Biosciences) and data was analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Li G., Liu H., Feng R., Kang T.S., Wang W., Ko C.N., Wong C.Y., Ye M., Ma D.L., Wan J.B, & Leung C.H. (2021). A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1–Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury. Redox Biology, 48, 102129.

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Multi-modal Immune Cell Profiling

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For surface staining, the cell suspension was incubated with fluorescently labeled antibody at room temperature for 20 minutes. For CD206 staining, the samples were fixed and permeabilised with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences, US), and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. For intracellular TNF-α staining, the samples were stimulated by LPS (100 ng/ml, Beyotime) for 4 h, fixed and permeabilised, and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. Bacteria were stained using the BacLight™ Red kit (Invitrogen, B-35001). miRNA was labelled with fluorescein amidites (FAM). The monoclonal antibodies used were as follows: APC-Cy7-anti-Mouse F4/80, Pacific Blue-anti-Mouse CD11b, FITC-anti-Mouse CD11c, APC-anti-Mouse CD206, APC-Cy7-anti-Mouse TCRβ, APC-anti-Mouse NK1.1, PerCP-Cy5-5-anti-Mouse CD45, PE-Cy7-anti-Mouse CD3, PE-anti-Mouse CD4, FITC-anti-Mouse CD8, APC-anti-Mouse TNF-α, all purchased from Biolegend; FITC-anti-Human CD86 (20 μl/ test), APC-anti-Human CD206 (20 μl/test), purchased from BD Pharmingen. Flow cytometry or Image Stream was conducted using a BD FACS CantoII or Millipore ISX with fluorochrome-conjugated cells, and the data was analysed using FlowJo software version 10.4 or IDEAS version 6.0.

Wang Y., Li Y., Lv L., Zhu L., Hong L., Wang X., Zhang Y., Wang X, & Diao H. (2024). Faecal hsa-miR-7704 inhibits the growth and adhesion of Bifidobacterium longum by suppressing ProB and aggravates hepatic encephalopathy. NPJ Biofilms and Microbiomes, 10, 13.

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Thymus and Spleen Cell Isolation

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The thymus and spleen were dissected from mice and then ground in cold PBS. The cell suspension was filtered through a 40 μm strainer. Red blood cells were lysed with red blood cell lysis buffer (70 (link)). The rest of the cells were analyzed by FACS for cell percentages or for cell culture. For thymocyte subpopulation assays, the cells were stained with the following surface antibodies in flow cytometry staining buffer (486.5 ml of 1× PBS, 12.5 ml of goat serum, and 1 ml of 0.5 M EDTA): PE anti-mouse CD127 (BioLegend), APC anti-mouse CD25 (BioLegend), FITC anti-mouse CD4 (BioLegend), PE-Cy5.5 anti-mouse CD8 (BioLegend), PE-Cy7 anti-mouse CD3 (BioLegend), and APC-Cy7 anti-mouse CD45 (BioLegend). The cells were incubated with the antibodies above in the tubes on ice for 45 min. The different subpopulations were counted and collected by FACS sorting (BD FACSAria III).

Zheng Y.Y., Wang Y., Chen X., Wei L.S., Wang H., Tao T., Zhou Y.W., Jiang Z.H., Qiu T.T., Sun Z.Y., Sun J., Wang P., Zhao W., Li Y.Q., Chen H.Q., Zhu M.S, & Zhang X.N. (2021). The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion. The Journal of Biological Chemistry, 298(1), 101516.

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Immunophenotyping and Functional Profiling of DCs

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DCs were sorted after isolation from spleen or colon from propyzamide- or vehicle-treated mice as described above. Cell suspensions were stained with FITC anti-mouse-NK1.1 (108706, BioLegend), CD64 (139316, BioLegend), Ly6G (127606, BioLegend), Ly6C (128006, BioLegend), B220 (553087, BD Bioscience) F/480 (11-4801-85, Thermo Fisher Scientific), PECy7 or PE-Dazzle594 anti-mouse-CD11c (N418, BioLegend) or PECy7-anti mouse CD3 (100220 and 117348, BioLegend) antibodies. After 30 min on ice, cells were washed and sorted on the FACS Aria Ilu (BD Biosciences) system. T cells were used to reconstituted Rag1-deficient mice as described below. DCs were pulsed with 10 μg ml⁻¹ of OVA 323-339 (SP-51023-1, Genemed) for 6 h, washed and co-cultured with naive CD4⁺ T cells isolated from spleens from B6.Cg-Tg(TcraTcrb)425Cbn/J OT-II mice using magnetic beads (130-104-453, Miltenyi). After 48 h, cells were restimulated and stained as described below.

Sanmarco L.M., Chao C.C., Wang Y.C., Kenison J.E., Li Z., Rone J.M., Rejano-Gordillo C.M., Polonio C.M., Gutierrez-Vazquez C., Piester G., Plasencia A., Li L., Giovannoni F., Lee H.G., Akl C.F., Wheeler M.A., Mascanfroni I., Jaronen M., Alsuwailm M., Hewson P., Yeste A., Andersen B.M., Franks D.G., Huang C.J., Ekwudo M., Tjon E.C., Rothhammer V., Takenaka M., de Lima K.A., Linnerbauer M., Guo L., Covacu R., Queva H., Fonseca-Castro P.H., Bladi M.A., Cox L.M., Hodgetts K.J., Hahn M.E., Mildner A., Korzenik J., Hauser R., Snapper S.B, & Quintana F.J. (2022). Identification of environmental factors that promote intestinal inflammation. Nature, 611(7937), 801-809.

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Tumor Immune Profiling by Flow Cytometry

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Mice were sacrificed and tumors were harvested. 0.3 mg tumor tissue was isolated and added into a solution of collagenase (400 units per mL), DNase 1 (100 μg mL⁻¹), and hyaluronidase (0.04 units per mL). The mixture was shaked under 37 °C for 1 h. After, the mixture was passed through a 70 μm nylon cell strainer. The sample was washed 3 times with PBS (with 2% FBS) and then resuspended in 1 × 10⁶ cells per mL for flow cytometry analysis. Antibody (AF488-anti mouse CD45, PE/Cy7 anti-mouse CD3, APC anti-mouse CD4, BV510-anti mouse CD8, PE anti-mouse FOXP3 and PE anti-mouse IFN-γ, all purchased from BioLegend) was added following the antibody manufacturer's recommendations and incubated for 30 min on ice. Cells were analyzed on BD FACS Celesta, and data were analyzed using FlowJo 10. Cellular events were first gated by forward and then by side scatter characteristics. The cell counts for the CD45⁺, CD45⁺CD3⁺, CD45⁺CD3⁺CD4⁺, CD45⁺CD3⁺CD4⁺FOXP3⁺ CD45⁺CD3⁺CD8⁺ and CD45⁺CD3⁺CD8⁺IFN-γ⁺ T lymphocytes were assessed. For quantification, the proportion of each immune subpopulation was determined by flow cytometry. This number was then multiplied by the total number of cells in the tumor, which had been determined before flow cytometry by counting with a hemocytometer. To determine cell density, cell counts were normalized to tumor weight (cell number per gram tumor).

Miao Q., Zhang W., Zhang K., Li H., Zhu J, & Jiang S. (2021). Rational design of a potent macrocyclic peptide inhibitor targeting the PD-1/PD-L1 protein–protein interaction. RSC Advances, 11(38), 23270-23279.

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Lycium barbarum Polysaccharide Immunomodulation

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LBP was prepared from Lycium barbarum by our laboratory as described previously [23 (link)]. The total sugar and protein content was 70.13% and 19.30%, respectively. The fractions with molecular weight range from 40 kDa to 350 kDa were prepared and used in this study. Mouse 1x lymphocyte separation medium was purchased from Dakewe Biotech Co. Ltd. (Shenzhen, China). PE/CY7-anti-mouse CD3, FITC-anti-mouse CD4, PE-anti-mouse PD-1, PE/CY5-anti-mouse CD25, and purified CD8 antibody were purchased from BioLegend. Purified CD3 antibody was purchased from Affinity Bioscience. Propidium iodide (PI), collagenase type IV, and DNase I were purchased from Sigma. Mouse IL-10 and mouse TGF-β1 ELISA kit were purchased from MULTI SCIENCE (Hangzhou, China).

Deng X., Luo S., Luo X., Hu M., Ma F., Wang Y., Lai X, & Zhou L. (2018). Polysaccharides from Chinese Herbal Lycium barbarum Induced Systemic and Local Immune Responses in H22 Tumor-Bearing Mice. Journal of Immunology Research, 2018, 3431782.

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Lymphocyte Immunophenotyping by Flow Cytometry

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The lymphocytes were isolated as mentioned in Section 4.5. After counting and washing with fresh sterile PBS (with 5% FBS, 500× g, 5 min) three times, the pelleted cells were re-suspended to 1 × 10⁷ cell/mL in PBS. The cell samples were divided into two tubes, 100 μL each, and antigens were added: PE/Cy7-anti-mouse CD3 (0.5 μg, the same below), FITC-anti-mouse CD4, PE-anti-mouse CD8a, and FITC-anti-mouse CD19, respectively (all Biolegend, San Diego, CA, USA) [41 ]. After mixing and incubation in dark at 4 °C for 30 min, the cells were washed with 900 μL cold-PBS twice, centrifuged (500× g, 5 min), and re-suspended with 300 μL PBS. The stained cells were analyzed with BD FACS Verse flow cytometer with FACSuite software (Becton Dickinson). Lymphocyte populations were determined as the percentages of T cells (CD3⁺, CD19⁻) and B cells (CD3⁻, CD19⁺) among leukocytes. Subpopulations of helper T cells and cytotoxic T cells are presented as the percentage of CD4⁺CD8⁻ and CD4⁻CD8⁺ cells among CD3⁺-expressing cells.

Zou Y.F., Zhang Y.Y., Fu Y.P., Inngjerdingen K.T., Paulsen B.S., Feng B., Zhu Z.K., Li L.X., Jia R.Y., Huang C., Song X., Lv C., Ye G., Liang X.X., He C.L., Yin L.Z, & Yin Z.Q. (2019). A Polysaccharide Isolated from Codonopsis pilosula with Immunomodulation Effects Both In Vitro and In Vivo. Molecules, 24(20), 3632.

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