Anti mouse cd3 clone 17a2

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The Anti-mouse CD3 (clone 17A2) is a laboratory reagent used for the detection and analysis of the CD3 antigen expressed on T cells in mouse samples. It functions as a tool for immunophenotyping and flow cytometry applications.

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Anti-CD3 (468 citations) Anti-mouse CD3 (46 citations) CD3 (17A2; (30 citations) CD3 (clone 17A2, (24 citations) Anti-CD3 (17A2, (22 citations) Clone 17A2 (20 citations) Anti-CD3 (clone 17A2, (19 citations) Anti-mouse CD3 (17A2, (5 citations) FITC anti‐mouse CD3 (clone 17A2, (2 citations) PE anti-mouse CD3 (clone 17A2), (2 citations)

5 protocols using anti mouse cd3 clone 17a2

Immunoblotting and Flow Cytometry Protocols

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For immunoblotting, these antibodies are used: rabbit anti-STAT1 (CST, #14994), rabbit anti–phospho-STAT1 (CST, #7649), rabbit anti-EZH2 (CST, #5246), mouse anti–MHC-I class I (Santa Cruz Biotechnology, #sc-55582), rabbit anti-IFNGR1 (Millipore, #MABF753), and mouse anti–β-actin (Sigma-Aldrich, #A5441).
For flow cytometry, the following fluorochrome-conjugated antibodies are used: anti-mouse H-2K^b/H-2D^b (clone 28-8-6, BioLegend), anti-mouse H-2Kb bound to SIINFEKL (clone 25-D1.16, BioLegend), anti-mouse CD3 (clone 17A2, BioLegend), anti-mouse CD28 (clone 37.51, BioLegend), anti-mouse granzyme B (clone GB11, BioLegend), anti-mouse B220 (clone RA3-6B2, BioLegend), anti-mouse CD49b (clone DX5, BioLegend), anti-mouse Gr-1 (clone RB6-8C5, BioLegend), anti-mouse MHC-II (clone M5/114.15.2, BioLegend), anti-mouse F4/80 (clone BM8, BioLegend), anti-mouse IFN-γ (XMG1.2, eBioscience), anti-mouse CD11b (M1/70, eBioscience), anti-mouse NK1.1 (clone PK136, BD), anti-mouse CD103 (clone M290, BD), anti-mouse CD206 (clone MR5D3, BD), anti-mouse CD24 (clone M1/69, BD), anti-mouse CD4 (clone GK1.5, BD), anti-mouse CD45 (clone 30-F11, BD), anti-mouse CD8a (clone 53-6.7, BD), anti-mouse Foxp3 (clone MF23, BD), anti-mouse γδ TCR (clone GL3, BD), anti-human CD3 (SK7, BioLegend), anti-human CD4 (SK3, BioLegend), anti-human CD8 (SK1, BioLegend), and anti-human 137(4-1BB) (4B4-1, BioLegend).

Guo W., Wang Y., Yang M., Wang Z., Wang Y., Chaurasia S., Wu Z., Zhang M., Yadav G.S., Rathod S., Concha-Benavente F., Fernandez C., Li S., Xie W., Ferris R.L., Kammula U.S., Lu B, & Yang D. (2021). LincRNA-immunity landscape analysis identifies EPIC1 as a regulator of tumor immune evasion and immunotherapy resistance. Science Advances, 7(7), eabb3555.

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Maternal CD8+ T Cell Analysis

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Flow cytometry was used to analyze CD8⁺ T cells in the maternal blood and placenta. Isolated lymphocytes were stained with surface markers in 2% fetal bovine serum in PBS for 30 minutes at 4°C in the dark. Antibodies used were anti-mouse CD45⁺ (clone 30-F11, ThermoFischer Scientific, Waltham, MA, USA), anti-mouse CD3⁺ (clone 17A2, BioLegend, San Diego, CA, USA), anti-mouse CD4⁺ (clone RM4-5, BD, Franklin Lakes, NJ, USA) and anti-mouse CD8α or its isotope (clone 2.43, TONBO Biosciences, San Diego, CA, USA). For blocking experiments, cells were incubated with unlabeled anti-CD8⁺ antibodies (clones YTS169.9 and 53-6.72, Bio X Cell, West Lebanon, NH, USA) in fluorescence activated cell sorting (FACS) buffer for 20 minutes at 4°C in the dark prior to surface marker staining. Data were acquired on a BD FACSCalibur^TM flow cytometer with Cellquest software (BD Bioscience, San Jose, CA, USA) and analyzed with FlowJo version 10.1 (FlowJo, LLC, Ashland, OR, USA).

Lei J., Xie L., Zhao H., Gard C., Clemens J.L., McLane M.W., Feller M.C., Ozen M., Novak C., Alshehri W., Alhejaily N., Shabi Y., Rosenzweig J.M., Facciabene A, & Burd I. (2017). Maternal CD8+ T cell depletion alleviates intrauterine inflammation-induced perinatal brain injury. American journal of reproductive immunology (New York, N.Y. : 1989), 79(5), e12798.

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Evaluating VISTA Protein Modulation of CD8+ T Cells

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CD8⁺ T cells were isolated as described above, and a density of 1 × 10⁵ cells/well were plated into the 96-well flat-bottom plates coated with anti-mouse CD3 (clone 17A2, Biolegend) at 2.5 μg/mL together with 5 μg/mL of murine VISTA-ECD protein. The compound 6809-0223 diluted into indicated concentrations was added to the culture medium. The cytokine levels in the cell supernatant collected at 48 h were analyzed using a BD Cytometric Bead Array (CBA) kit according to the manufacturer’s instructions.

Li T.T., Jiang J.W., Qie C.X., Xuan C.X., Hu X.L., Liu W.M., Chen W.T, & Liu J. (2021). Identification of active small-molecule modulators targeting the novel immune checkpoint VISTA. BMC Immunology, 22, 55.

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T Cell Activation and Culture

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Cells were stained with 5 μM of CellTrace Violet (Thermo Fisher Scientific) for 5 min at room temperature. A half volume of FBS was then added for 5 min at room temperature before washing with media. Mouse splenocytes were activated using 1 μg/ml of plate-bound anti-mouse CD3 (clone 17A2; BioLegend) and 1 μg/ml soluble anti-mouse CD28 (clone 37.51; BioLegend) in cRPMI. Human PBMCs were stimulated with 1 µg/ml Ultra-LEAF purified anti-CD3 (clone HIT3α; BioLegend) and 1 µg/ml Ultra-LEAF purified anti-CD28 (clone CD28.2; BioLegend) soluble antibodies in supplemented RPMI-1640 (10% FBS, 100 U/ml penicillin and streptomycin, 2 mM Glutamax, and 20 mM HEPES). To culture blasting T cells, activated T cells were washed and then cultured in a medium supplemented with 100 U/ml IL-2.

Yao Y., Du Jiang P., Chao B.N., Cagdas D., Kubo S., Balasubramaniyam A., Zhang Y., Shadur B., NaserEddin A., Folio L.R., Schwarz B., Bohrnsen E., Zheng L., Lynberg M., Gottlieb S., Leney-Greene M.A., Park A.Y., Tezcan I., Akdogan A., Gocmen R., Onder S., Rosenberg A., Soilleux E.J., Johnson E., Jackson P.K., Demeter J., Chauvin S.D., Paul F., Selbach M., Bulut H., Clatworthy M.R., Tuong Z.K., Zhang H., Stewart B.J., Bosio C.M., Stepensky P., Clare S., Ganesan S., Pascall J.C., Daumke O., Butcher G.W., McMichael A.J., Simon A.K, & Lenardo M.J. (2022). GIMAP6 regulates autophagy, immune competence, and inflammation in mice and humans. The Journal of Experimental Medicine, 219(6), e20201405.

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Murine Immune Cell Profiling

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Murine samples of spleen tissues, lymph nodes and tumors were collected and prepared as previously described (23) . Cells were stained with the following antibodies: antimouse CD3 (clone 17A2, BioLegend, Cat#100221, RRID:AB_2057374), antimouse CD4 (clone GK1.5, Miltenyi Biotec, Cat#130-121-131, RRID:AB_2752219), antimouse CD8a (clone 53-6.7, BioLegend, Cat#100712, RRID:AB_312751), antimouse CD45 (clone 30-F11, BioLegend, Cat#103108, RRID:AB_312973, BD Biosciences, Cat#557659, RRID:AB_396774), antimouse F4/80 (clone BM8, BioLegend, Cat#123115, RRID:AB_893493), antimouse CD11b (clone M1/70, Miltenyi Biotec, Cat# 130-097-585, RRID:AB_2660136), antimouse Gr-1 (clone RB6-8C5, TONBO, San Diego, CA), antimouse Ly6C (clone HK1.4, BioLegend, Cat#128006, RRID:AB_1186135). Data was acquired using MACS Quant flow cytometer (Miltenyl Biotec, Bergisch Galdbach, Germany) and analyzed using FlowJo software (FlowJo, RRID:SCR_008520).

Mise Y., Hamanishi J., Daikoku T., Takamatsu S., Miyamoto T., Taki M., Yamanoi K., Yamaguchi K., Ukita M., Horikawa N., Abiko K., Murakami R., Furutake Y., Hosoe Y., Terakawa J., Kagabu M., Sugai T., Osakabe M., Fujiwara H., Matsumura N., Mandai M, & Baba T. (2022). Immunosuppressive tumor microenvironment in uterine serous carcinoma via CCL7 signal with myeloid-derived suppressor cells. Carcinogenesis, 43(7).

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