Anti tim 3 pe cy7

Manufactured by BioLegend

Sourced in United States

Anti-Tim-3-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the human T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). Tim-3 is an inhibitory receptor expressed on T cells, NK cells, and other immune cells. The PE-Cy7 fluorescent label allows for the detection and analysis of Tim-3-expressing cells using flow cytometry.

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5 protocols using anti tim 3 pe cy7

Profiling Exhausted T Cell Markers

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The expression of exhausted and cytotoxic markers of T cell was analyzed by flow cytometry. After 5 days of co-culture, cells were suspended in PBS containing 2% FBS and incubated according to the manufacturer’s instructions with the following fluorochrome-labeled antibodies: anti-CD8-APC-A700 (#B49181, Beckman Coulter), anti-CD4-APC (#IM2468, Beckman Coulter), anti-TIGIT-PE-Cy7 (#372714, Biolegend), anti-CTLA-4-BV785 (#369624, Biolegend), anti-Tim-3-PE-Cy7 (#345052, Biolegend), anti-LAG-3-BV605 (#369324, Biolegend), anti-PD-1-BV510 (#367424, Biolegend), anti-Granzyme B-PE (#372208, Biolegend), anti-IFN-γ-FITC (#IM2716U, Beckman Coulter). Cells were stained with fluorochrome-conjugated antibodies for 30 min at room temperature in the dark. For intracellular staining, surface-stained cells were fixed and permeabilized using PerFix-nc Kit (#B31168, Beckman Coulter) according to the manufacturer's instructions. Flow cytometry analyses were performed on DxFlex system (Beckman Coulter) and data were analyzed using FlowJo software (v10.5.3).

Chen M., Wan Y., Li X., Xiang J., Chen X., Jiang J., Han X., Zhong L., Xiao F., Liu J., Huang H., Li H., Liu J, & Hou J. (2023). Dynamic single-cell RNA-seq analysis reveals distinct tumor program associated with microenvironmental remodeling and drug sensitivity in multiple myeloma. Cell & Bioscience, 13, 19.

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Comprehensive Pancreatic Immune Profiling

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Pancreas and pancreatic tumor single-cell suspensions were washed first with PBS and then Fc receptors were blocked for 10 min at 4°C. For pancreatic myeloid cells, single-cell suspensions were stained with viability dye-APC eFluor-780 (eBioscience), anti-CD45-PerCP-Cy5.5, anti-CD11b-PE-Cy7, anti-F4/80-FITC, anti-Ly6C-AlexaFluor 700, and anti-Ly6G-APC (BioLegend). For T cells, cells were stained with viability dye-APC eFluor 780, anti-CD3-PE-Cy5, anti-CD4-APC, anti-CD8-PerCp-Cy5.5, anti-CD69-AlexaFluor 700, anti-TIM-3-PE-Cy7, anti-LAG-3-PE-Dazzle 594, and anti-PD-1-APC (BioLegend). Cells were incubated at 4°C for 30 min, washed with cold PBS, filtered, and collected using CyTEK Aurora Northern lights 2 laser flow cytometer (CyTEKbio). All flow cytometry data were analyzed using FlowJo software (BD).

Woeste M.R., Shrestha R., Geller A.E., Li S., Montoya-Durango D., Ding C., Hu X., Li H., Puckett A., Mitchell R.A., Hayat T., Tan M., Li Y., McMasters K.M., Martin R.C, & Yan J. (2023). Irreversible electroporation augments β-glucan induced trained innate immunity for the treatment of pancreatic ductal adenocarcinoma. Journal for Immunotherapy of Cancer, 11(4), e006221.

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PD-1/PD-L1/PD-L2 Expression Profiling

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PD-1 ligand expression was determined using anti-PD-L1-PE or PD-L2-PE, combined with HLA-DR-perCP (BD, Franklin Lakes, NJ). PD-1 expression was determined using anti-PD-1-PE/isotype control (BioLegend, San Diego, CA), in combination with anti-CD3-PB, CD45RA-V500 and HLA-DR-APC-Cy7 (BD). At day 5 post-infection eFluor670^highEGFP⁻CD45RA⁻ T-cells were sorted into PD-1^high and PD-1^low/− populations. Some donors were labelled with both anti-PD-1-PE and anti-Tim-3-PE-Cy7 (BioLegend), and cells positive for both or either IC (PD-1/Tim-3^high), or cells negative for both IC (PD-1^low/−Tim-3^low/−) were sorted.

EVANS V.A., VAN DER SLUIS R.M., SOLOMON A., DANTANARAYANA A., McNEIL C., GARSIA R., PALMER S., FROMENTIN R., CHOMONT N., SÉKALY R.P., CAMERON P.U, & LEWIN S.R. (2018). PD-1 contributes to the establishment and maintenance of HIV-1 latency. AIDS (London, England), 32(11), 1491-1497.

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Multiparameter Immune Cell Profiling

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Anti–CD3ε-PerCP (100326), anti–CD8a-AF700 (100730), anti–CD8a-APC (100712), anti–PD-1-PE/DZ594 (109116), anti–Ki67-FITC (652410), anti–TCF1-PE (655208), anti–IFN-γ-BV421 (505830), anti–IFN-γ-PerCp/Cy5.5 (505822), anti–TNF-α-APC/Cy7 (506344), anti–TIM-3-PE/Cy7 (119716), anti–CD45-BV510 (103138), anti–Ly108 (Slamf6)-PE (134606), anti–PD-1-FITC (135214), anti–CD45.1-PE (110708), anti–CD45.2-AF488 (109816), purified anti-mouse CD3ε (100340), and purified anti-mouse CD28 (102116) were obtained from Biolegend. Anti–CD8-FITC (D271-4) and tetramer-SIINFEKL-APC (TS-5001-2c) were purchased from MBL. Cell Stimulation Cocktail Plus Protein Transport Inhibitors and FOXP3/TF Staining Buffer Set were bought from eBioscience. Naive CD8a⁺ T Cell Isolation and Tumor Dissociation Kits were obtained from Miltenyi Biotec.

Li X., Li Y., Dong L., Chang Y., Zhang X., Wang C., Chen M., Bo X., Chen H., Han W, & Nie J. (2023). Decitabine priming increases anti–PD-1 antitumor efficacy by promoting CD8+ progenitor exhausted T cell expansion in tumor models. The Journal of Clinical Investigation, 133(7), e165673.

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Multicolor Flow Cytometry Antibody Panel

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The following antibodies were purchased from BioLegend (San Diego, CA, USA): anti-CD4-PE-CY7, anti-CD8-APC, anti-CD3-FITC, anti-CD45-PE, anti-Gr-1-APC, anti-CD11b-PE, anti-F4/80-FITC, anti-CD206-APC, anti-CD86-PE-CY7, anti-CD4-FITC, anti-CD25-APC, anti-Foxp3-PE, anti-PD-1-FITC, anti-Tim3-PE-CY7, TruStain FcX PLUS (anti-mouse CD16/32), and isotype control. Antibody concentrations were used according to the manufacturer’s instructions.

Geng F., Dong L., Bao X., Guo Q., Guo J., Zhou Y., Yu B., Wu H., Wu J., Zhang H., Yu X, & Kong W. (2022). CAFs/tumor cells co-targeting DNA vaccine in combination with low-dose gemcitabine for the treatment of Panc02 murine pancreatic cancer. Molecular Therapy Oncolytics, 26, 304-313.

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