Apc cy7 anti mouse cd3

Manufactured by BioLegend

Sourced in United States

The APC/Cy7 anti-mouse CD3 is a fluorescently conjugated antibody that binds to the CD3 antigen on mouse T cells. It can be used to detect and quantify T cells in flow cytometry applications.

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Lab products found in correlation

Brefeldin a, by Thermo Fisher Scientific (2 mentions) Anti mouse cd45 vioblue, by Miltenyi Biotec (1 mentions) Anti mouse cd11b bv605, by BioLegend (1 mentions) Anti mouse cd44 af700, by BD (1 mentions) Viacount reagent, by Merck Group (1 mentions) Apc anti mouse cd4, by BioLegend (1 mentions) Anti mouse cd8 pe cy7, by BioLegend (1 mentions) Anti mouse ifn γ pe, by Thermo Fisher Scientific (1 mentions) Monensin, by BioLegend (1 mentions) Guava easycyte 8ht, by Merck Group (1 mentions) Lsr 2, by BD (1 mentions) Lymphocyte separation medium, by Dakewe (1 mentions) Monensin, by MultiSciences Biotech (1 mentions) Apc anti mouse cd49b, by BioLegend (1 mentions) Apc anti mouse cd62l, by BioLegend (1 mentions) Fitc anti mouse cd8, by BD (1 mentions) Anti mouse cd11b fitc, by BD (1 mentions) Fitc anti mouse cd4, by BD (1 mentions) Apc anti mouse cd8, by BioLegend (1 mentions) Pe anti mouse cd44, by BD (1 mentions)

8 protocols using apc cy7 anti mouse cd3

Identification of Liver-Infiltrating T Cells

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Purity of OT-I CD8⁺ T cells were verified by flowcytometry, using anti-mouse V alpha 2 TCR‑FITC (eBioscience, San Diego, CA, USA) and anti-mouse Vβ 5.1, 5.2 TCR-PE (BD Bioscience, Heidelberg, Germany) antibodies. A CD16/CD32 anti‑mouse antibody, incubated for 15 min, was used to block Fc receptor binding, before the α-CD8α-APC antibody for T cells was added on ice. Surface expression of PD-L1 and PD-L2 on the surface of primary hepatocytes was determined by FACS analysis, using α-PD-L1-PE or α-PD-L2-PE. Immune cells were excluded by CD45-FITC staining, consequently analyzing CD45^- cells only.
To identify regulatory T cells (Tregs) in liver single cell preparations, cells were stained for anti-mouse CD45-Vioblue (Miltenyi Biotec, Bergisch-Gladbach, Germany), anti-mouse CD3-APC-Cy7 (BioLegend, Eching, Germany) , anti-mouse CD4-BV711 (BD Horizon, Heidelberg, Germany), anti-mouse CD11b-BV605 (BioLegend), anti-mouse CD25-PE-Cy7 (BD Pharmingen, Heidelberg, Germany), anti-mouse GITR-FITC (BioLegend), and anti-mouse CD44-AF700 (BD Pharmingen) in parallel. CD4⁺ T cells showing expression of CD25, GITR, combined with CD44 were judged as Tregs. FACS measurements were performed by a FACS Fortessa LSR and data analysis was done with the FlowJo software.

von Knethen A., Schäfer A., Kuchler L., Knape T., Christen U., Hintermann E., Fißlthaler B., Schröder K., Brandes R.P., Genz B., Abshagen K., Pützer B.M., Sha L.K., Weigert A., Syed S.N., Schulz M., Shah A.M., Ernst A., Putyrski M., Finkelmeier F., Pesic M., Greten F., Hogardt M., Kempf V.A., Gunne S., Parnham M.J, & Brüne B. (2019). Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis. Theranostics, 9(7), 2003-2016.

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Splenocyte IFN-γ Secretion Assay

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Mice were immunized as mentioned above. PBS-treated mice were used as the control. Spleens were excised from the mice 14 days after the first immunization. Splenocytes were separated and counted on a Guava easyCyte 8HT (Merck Millipore, Billerica, Massachusetts, USA) with Viacount reagent (Merck Millipore, Billerica, Massachusetts, USA). The densities of the splenocytes were adjusted to 2.0 × 10⁶ cells/ml in complete RPMI 1640 containing 10% FBS. Splenocytes from each mouse were added to 2 wells of a 24-well plate (4.0 × 10⁶ cells per well). For the tfRFP-stimulated groups, 50 μg/ml tfRFP was added to the medium. An equal volume of PBS was added to the control groups. The splenocytes were cultured at 37ºC for 3 days. IFN-γ secretion was inhibited by monensin (Biolegend, San Dieg, California, USA) for the last 4 h of the culture. Finally, collected cells were fixed and stained with anti-mouse CD3 APC/Cy7 (Biolegend, San Dieg, California, USA), anti-mouse CD4 APC (Biolegend, San Dieg, California, USA), anti-mouse CD8 PE/Cy7 (Biolegend, San Dieg, California, USA), and anti-mouse IFN-γ PE (eBioscience, San Diego, California, USA) for flow cytometric analysis. A rat anti-mouse IgG1 kappa PE antibody (eBioscience, San Diego, California, USA) was used as an isotype control.

Yang F., Liu S., Liu X., Liu L., Luo M., Qi S., Xu G., Qiao S., Lv X., Li X., Fu L., Luo Q, & Zhang Z. (2016). In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity. Theranostics, 6(9), 1453-1466.

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Flow Cytometric Analysis of Splenic Lymphocytes

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Splenic lymphocytes were harvested by gradient centrifugation using the lymphocyte separation medium (Dakewe Biotech, China) at room temperature and washed twice with PBS. To determine cytokine expression, cells were stimulated with phorbol12-myristate 13-acetate (PMA)/ionomycin and monensin (Multiscience, China) for 6 h. For flow cytometric analysis, cells were suspended in staining buffer and incubated for 15 min at room temperature by labeling APC/Cy7 anti-mouse CD3 (Clone: 17A2, Biolegend, USA), Brilliant Violet 421^TM anti-mouse CD8a (Clone: 53-67, Biolegend, USA), and PE anti-mouse CCL5 (Clone: 2E9, Biolegend, USA). The cells were finally resuspended in 500 μL of PBS and subjected to flow cytometry (BD LSR II).

Wu Q., Hu X., Zhang X., Kong D., Yang Z., Li G., Gu Z., Zhang Q., Wan D., Cheng S., Liu B., Zhang K, & Zhang W. (2022). Single-cell transcriptomics of peripheral blood reveals anti-tumor systemic immunity induced by oncolytic virotherapy. Theranostics, 12(17), 7371-7389.

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Comprehensive Murine Immune Cell Profiling

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The following antibodies were used for FACS staining: APC/Cy7 anti-mouse CD3 (100221, Biolegend, San Diego, CA, USA), FITC anti-mouse CD4 (553729, BD Bioscience, San Jose, CA, USA), FITC anti-mouse CD8 (553031, BD Bioscience), APC anti-mouse CD8 (100711, Biolegend), APC anti-mouse Foxp3 (4331294, Invitrogen, Waltham, MA, USA), APC anti-mouse CD62L (104411, Biolegend), PE anti-mouse CD44 (553134, BD Bioscience), APC anti-mouse CD49b (108909, Biolegend), FITC anti-mouse CD11b (557396, BD Bioscience), H-2K^b/OVA (SIINFEKL)-Tetramer/PE was kindly provided by Prof. Eui-Cheol Shin (KAIST, Daejeon, Korea).

Do-Thi V.A., Lee H., Jeong H.J., Lee J.O, & Kim Y.S. (2021). Protective and Therapeutic Effects of an IL-15:IL-15Rα-Secreting Cell-Based Cancer Vaccine Using a Baculovirus System. Cancers, 13(16), 4039.

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Comprehensive Antibody Panel for Cellular Signaling

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The following antibodies were used: anti-B7-H3 (Proteintech #66 481–1-Ig); antiphospho-p38 MAPK (Thr180/Tyr182) (CST #4511); anti-p38 (CST #9212); antiphospho-eIF4E (S209) (HUABIO #ET1608-66); anti-eIF4E (Proteintech #66 655–1-Ig); anti-HER2 (Proteintech #18 299–1-AP); anti-α-tubulin (Beyotime Biotechnology #AF5012); anti-β-actin (Proteintech #6009–1-Ig); anti-PDL1 (CSB-MA878942A1 m); anti-EGFR (ZENBIO# 201012); HRP conjugated goat antirabbit IgG goat polyclonal antibody (HUABIO #HA1001); and HRP-conjugated goat antimouse IgG goat polyclonal antibody. Antibodies for flow cytometry, including PerCP-Cy5.5-antimouse CD45, FITC-antimouse CD11B, APC-antimouse B7-H3, APC-Cy7-antimouse CD3, FITC-antimouse CD4, BV510-antimouse CD8, APC-antimouse F4-80, BV421-antimouse CD86, PE-antimouse CD206, and PE-antihuman B7-H3, were all purchased from Biolegend.
Cells were treated with the following reagents: SB203580 (Beyotime Biotechnology# S1863); recombinant human TNF-alpha protein (Sinobiological# 10602-HNAE); and lipopolysaccharide (LPS) (Biosharp#BS904).

Zhao S., Wang Y., Yang N., Mu M., Wu Z., Li H., Tang X., Zhong K., Zhang Z., Huang C., Cao T., Zheng M., Wang G., Nie C., Yang H., Guo G., Zhou L., Zheng X, & Tong A. (2022). Genome-scale CRISPR–Cas9 screen reveals novel regulators of B7-H3 in tumor cells. Journal for Immunotherapy of Cancer, 10(6), e004875.

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Cytokine Production in Peptide-Stimulated Lung Cells

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Isolated lung cells were stimulated with a peptide (A-NP_147–155 or B-NP_166–174) at 37°C for 5 h in the presence of brefeldin A (Thermo Fisher Scientific). They were blocked using a purified rat anti-mouse CD16/CD32 Fc blocker (BD Pharmingen, San Diego, CA, USA). Thereafter, the cell surface was stained with APC/Cy7 anti-mouse CD3 (Clone 17A2; BioLegend) and FITC anti-mouse CD8 (clone KT15; MBL Life Science, Woburn, MA, USA). After 30 min, they were intracellularly stained for cytokine production using a BD Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) in accordance with the manufacturer’s instructions. For IFN-γ and TNF-α measurements, APC anti-mouse IFN-γ (clone XMG1.2; BioLegend) and PE anti-mouse TNF-α (clone MP6-XT22; BioLegend) were added to the samples.

Kong H.J., Choi Y., Kim E.A, & Chang J. (2023). Vaccine Strategy That Enhances the Protective Efficacy of Systemic Immunization by Establishing Lung-Resident Memory CD8 T Cells Against Influenza Infection. Immune Network, 23(4), e32.

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Evaluating IFN-γ Response to Cas9 Delivery

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Mice were injected thrice with empty LNPs, sgRNA/Cas9 mRNA–encapsulated LNPs, and PBS. Twenty-four hours after the last injection, single-cell suspensions were prepared from mice spleens. Cells (1 × 10⁶) were cultured with medium alone (negative control), 2.5 μg of recombinant Cas9 (Cas9), or phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml) (positive control) for 18 hours. To measure the IFN-γ–producing cells, splenocytes were further treated with brefeldin A (eBioscience Inc., San Diego, CA, USA) for 5 hours. Cells were then permeabilized with fluorescence-activated cell sorting (FACS) buffer containing 0.5% saponin (Sigma-Aldrich, St. Louis, MO, USA) and stained with markers. The stained cells were acquired using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). The antibodies used were antigen-presenting cell (APC)/Cy7 anti-mouse CD3 (BioLegend, San Diego, CA, USA, 100706), fluorescein isothiocyanate (FITC) anti-mouse CD8 (BioLegend, 100222), and phycoerythrin (PE) anti-mouse IFN-γ (BioLegend, 505808).

Han J.P., Kim M., Choi B.S., Lee J.H., Lee G.S., Jeong M., Lee Y., Kim E.A., Oh H.K., Go N., Lee H., Lee K.J., Kim U.G., Lee J.Y., Kim S., Chang J., Lee H., Song D.W, & Yeom S.C. (2022). In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy. Science Advances, 8(3), eabj6901.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions were prepared from host spleen, lung, and colon by mechanical disruption and filtering using a metallic mesh. Live cells were quantified by using Trypan blue exclusion cell assay. The following fluorescent-conjugated antibodies were used for flow cytometry: APC/Cy7 anti-mouse CD3 (Biolegend, San Diego, CA), PE anti-mouse H-2K^b, APC anti-mouse CXCR3, PE anti-mouse CCR5, PE anti-mouse CCR7 (eBioscience, San Diego, CA). Samples were run using a four fixed-aligned laser flow cytometer and data were analyzed using BD FACS Diva^TM software (Becton Dickinson, Mountain View, CA) and FlowJo software (TreeStar, Ashland, OR).

Robles J.D., Liu Y.P., Cao J., Xiang Z., Cai Y., Manio M., Tang E.H, & Chan G.C. (2015). Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models. Experimental Hematology & Oncology, 4, 13.

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