Granzyme b apc

Manufactured by BioLegend

Granzyme B APC is a fluorochrome-conjugated antibody that binds to the serine protease Granzyme B, which is involved in the induction of apoptosis in target cells. The APC (Allophycocyanin) fluorochrome allows for detection and quantification of Granzyme B-expressing cells using flow cytometry.

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Granzyme B (61 citations) Granzyme B-APC-fire750 (2 citations) Anti-granzyme B-APC (2 citations)

8 protocols using granzyme b apc

Assessing Marburg Virus Antibody-Mediated NK Cell Responses

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Nunc Maxisorp Immuno plates (Thermo Fisher Scientific) were coated with 1 μg/mL recombinant soluble MARV-Angola GPdTM (Alpha Diagnostics) in PBS (50 ul/well). Serum samples were heat-inactivated at 56 °C for 30 min, diluted in DMEM and then mixed with the PBMCs isolated. The antibody-PBMC mixture was transferred to the coated plate and incubated for 24 h at 37 °C. The cells were then stained for NK cell immune responses utilizing Live/Dead-UV450, CD45-BV786 (BD Biosciences Cat# 563861, RRID:AB_2738454), CD3-FITC (BD Biosciences Cat# 556611, RRID:AB_396484), CD8-PeTexas Red (Beckman Coulter Cat# 6607123, RRID:AB_1575983), CD16-AF700 (BioLegend Cat# 302026, RRID:AB_2278418), CD20-BV421 (BioLegend Cat# 302330, RRID:AB_10965543), and CD107a-PE (BioLegend Cat# 328608, RRID:AB_1186040). Cells were fixed with 4% paraformaldehyde (PFA) and stained intracellularly with IFN-γ-PE-Cy7 (Thermo Fisher Scientific Cat# 25-7319-82, RRID:AB_469682) and Granzyme B-APC (BioLegend Cat# 372204, RRID:AB_2687028) diluted in Perm-Wash buffer (Biolegend). Sample acquisition was performed on a Cytoflex-S (Beckman Coulter, Brea, CA) and data analyzed in FlowJo V10.

O'Donnell K.L., Feldmann F., Kaza B., Clancy C.S., Hanley P.W., Fletcher P, & Marzi A. (2023). Rapid protection of nonhuman primates against Marburg virus disease using a single low-dose VSV-based vaccine. eBioMedicine, 89, 104463.

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Multi-parameter Immune Cell Profiling

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Cells were incubated 10 min with the TruStain fcX (clone 93) washed and incubated with the antibodies during 20 minutes followed by two washes with PBS. Monoclonal antibodies specific for mouse molecules were purchased from Biolegend: CD3-FITC (clone 17A2), CD3-APC (clone 17A2), CD3-PerCp/Cy5.5 (clone 17A2), CD8-Brillant Violet 421 (clone 53-6.7), CD45-PE (clone 30-F11), CD45-PErCP(clone 30-F11), CD45.1-PE/Cy7 (clone A20), CD45.1-FITC (clone A20), CD103-APC (clone 2E7), CD103-PerCP (clone 2E7), CD69-APC/Cy7 (clone H1.2F3), CD69-APC (clone H1.2F3), CD44-PerCP (clone IM7), IFN-γ-PE (clone XMG1.2), IFN-γ-APC (clone XMG1.2), TNF-α-APC/Cy7 (clone MP6-XT22), CD11b-FITC (clone M1/70), CD207-PE (clone 4C7), XCR1-APC (clone ZET), XCR1-PerCP-Cy5.5 (clone ZET), CD11c-PE/Cy7 (clone N418), MHCII-APC/Cy7 (clone M5/114.15.2), CD24-PerCP-Cy5.5 (clone M1/69), CD80-APC (clone 16-10A1), CD80-PE/Cy7 (clone 16-10A1), CD86 Brilliant Violet 421 (clone GL-1), CCR7-PE/Cy7 (clone 4B12), IL-2-PE/Cy7 (clone JES6-5H4) IL-12/23-APC (clone C15.6), granzyme B-APC (clone GB11) and viability dye Zombie Aqua (ref 423101). Samples were acquired in a FACSCanto II cytometer (BD Bioscience) and data were analyzed using FlowJo version X.0.7 (Tree Star, Inc.). Gating strategies for all flow cytometry experiments are shown in Supplementary Fig. 4.

Menares E., Gálvez-Cancino F., Cáceres-Morgado P., Ghorani E., López E., Díaz X., Saavedra-Almarza J., Figueroa D.A., Roa E., Quezada S.A, & Lladser A. (2019). Tissue-resident memory CD8+ T cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells. Nature Communications, 10, 4401.

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Comprehensive Immune Cell Analysis

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The following antibodies were used for flow cytometry analysis: CD45-FITC (Cat. # 553079; BD Biosciences), CD3-BUV395 (Cat. # 563565; BD Biosciences), CD4-BUV737 (Cat. # 612761; BD Biosciences), CD8-Percp-Cy5.5 (Cat. # 45-0081-82; eBioscience), CD44-BV711 (Cat. # 103057; Biolegend), CD335-PE/Dazzle594 (Cat. #137630; Biolegend), PD-1-PE (Cat. # 551892; BD Biosciences), Ki67*-BV605 (Cat. # 652413; Biolegend), Granzyme B*-APC (Cat. # 366408; Biolegend), IFN-γ*-BV421 (Cat. # 563376; BD Biosciences), CD11b-PE-Cy7 (Cat. # 101216; Biolegend), F4/80-BV510 (Cat. # 123135; Biolegend), CD206-AF700 (Cat. # 141734; Biolegend), I-A/I-E-BV786 (Cat. # 743875; BD Biosciences), and L/D-efluor780 (Cat. # 65-0865-18; eBioscience).

Zhang Y., Gabere M., Taylor M.A., Simoes C.C., Dumbauld C., Barro O., Tesfay M.Z., Graham A.L., Ferdous K.U., Savenka A.V., Chamcheu J.C., Washam C.L., Alkam D., Gies A., Byrum S.D., Conti M., Post S.R., Kelly T., Borad M.J., Cannon M.J., Basnakian A, & Nagalo B.M. (2022). Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy. Frontiers in Oncology, 12, 1042250.

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Functional Characterization of T Cell Subsets

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Total CD3+ T cells were cotransduced with CAR lentivirus and either pTRIP-SFFV-GFP or pTRIP-SFFV-GFP-RELA K5R for 4 d prior to staining. 0.15 million cells were treated with cell-stimulation cocktail and protein transport inhibitors (#15516286 or 15172069; Thermo Fisher Scientific) for 4 h. Cells were stained with Fixable Viability Dye eFluor 780 (#15383562; Thermo Fisher Scientific), CD4 BV510 (#562971; Thermo Fisher Scientific), and CD8a PE (#15506706; Thermo Fisher Scientific), and then fixed and permeabilized using the eBioscience FOXP3 transcription factor staining buffer set (#11500597; Thermo Fisher Scientific). Permeabilized cells were stained with antibodies against IFNγ PeCy7 (#13417646; Thermo Fisher Scientific) and Granzyme B APC (#515405; Biolegend). Samples were acquired on a NovoCyte (Agilent). Isotype controls were used to define gates.

Jeremiah N., Ferran H., Antoniadou K., De Azevedo K., Nikolic J., Maurin M., Benaroch P, & Manel N. (2023). RELA tunes innate-like interferon I/III responses in human T cells. The Journal of Experimental Medicine, 220(5), e20220666.

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Multicolor Flow Cytometry for Treg Immunophenotyping

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Multicolour flow cytometry was used to assess the immunophenotype of Tregs. Antibodies against the following molecules were provided by: CD3 BV650 (BD Biosciences), CD8 BV450 (BD Biosciences), CD4 APC-H7 (BD Biosciences), CD25 PerCPCy5.5 (BD Biosciences), CD73 eFluor 450 (eBioscience™), PD-1 BV 650 (Biolegend). Dead cells were stained by LIVE/DEAD^® Fixable Blue Dead Cell Stain Kit (Life Technology). For intracellular staining, cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience), and then stained with FoxP3 Alexa Fluor 488 (eBioscience), IL13-PE (eBioscience) and Granzyme B APC (BioLegend). Appropriate isotype controls were used to set the quadrants and to evaluate background staining. Cells were analysed using an LSRII flow cytometer with BD FACSDIVA software.

Faridar A., Thome A.D., Zhao W., Thonhoff J.R., Beers D.R., Pascual B., Masdeu J.C, & Appel S.H. (2020). Restoring regulatory T-cell dysfunction in Alzheimer’s disease through ex vivo expansion. Brain Communications, 2(2), fcaa112.

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Profiling Immune Cells by Flow Cytometry

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Monoclonal antibodies specific for human CD3-PeCy7, CD3-APC-Cy7 (Clone HIT3a), CD4-Alexa Fluor 488 (Clone OKT4), CD8-APC-Cy7, CD8-Brilliant Violet 421 (Clone RPA-T8), IgG1-PE (Clone HP6017), IgG1 Isotype-PE (Clone MOPC−21), 41BB-PE (Clone 4B4–1), PD−1-PeCy7, PD−1-Brilliant Violet 421 (Clone EH12.2H7), CD69-APC-Cy7 (Clone FN50), IFNγ-APC-Cy7 (Clone 4S.B3), TNFα-Alexa Fluor 488 (Clone Mab11), granzyme B-APC (Clone GB−11), perforin-PerCPCy5.5 (Clone B-D48), CEA-APC (Clone ASL−32), PD-L1-Brilliant Violet 421 (Clone 29E.2A3), TruStain fcX™ (Clone 93) and viability dye Zombie Aqua (ref 423,101), were purchased from Biolegend. Samples were analyzed in a FACSCanto II cytometer (BD Bioscience) and data were analyzed using FlowJo version X.0.7 (Tree Star, Inc.).

Lopez E., Hidalgo S., Roa E., Gómez J., Hermansen Truan C., Sanders E., Carrasco C., Pacheco R., Salazar-Onfray F., Varas-Godoy M., Borgna V, & Lladser A. (2023). Preclinical evaluation of chimeric antigen receptor T cells targeting the carcinoembryonic antigen as a potential immunotherapy for gallbladder cancer. Oncoimmunology, 12(1), 2225291.

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Analyzing Mouse Mammary Tumor Immune Cells

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Mouse mammary tumor nodes were excised and divided into two parts for histopathological analysis and flow cytometry analysis, respectively. Tumor tissues for flow cytometry were cut into small pieces and digested with 1 mg/mL collagenase type IV (07912; Stemcell) and 0.5 Kunits/ml DNase (D7073; Biotopped) for 0.5 h. Samples were then filtrated to single-cell suspension. Cells were stained with CD45 À PerCP-Cyanine5.5 (103132; Biolegend; 1:20), CD8-FITC (100706; Biolegend; 1:20), CD3-PE-Cyanine7 (100320; Biolegend; 1:20) and Granzyme B-APC (100706; Biolegend; 1:20). Subsequently, cells were analyzed by flow cytometry. The results were analyzed by FlowJo X.

Liu Y., Peng Y., Du W., Yu C., Peng Z., Qin L., Ma Y., Wu X., Peng Y., Cheng X., Xia L., Fa H., Wu Y., Sun L., Liu J., Liu Z., Shang Y., Wang S, & Liang J. (2023). PD-L1-mediated immune evasion in triple-negative breast cancer is linked to the loss of ZNF652. Cell reports, 42(11).

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Characterizing Murine T-cell Phenotypes

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T-cells collected from the spleen of wildtype BALB/c mice were stained with Live/Dead Fixable Yellow (ThermoFisher Scientific L34968) at a 1:1000 concentration for 30 min ice, rinsed with staining buffer and then blocked with CD16/32 FCR blocker at a 1:100 concentration (BioLegend, 101319) for 10 min on ice. Cells were then stained with antibodies specific for cell surface markers for 30 min on ice. The surface markers analyzed were: CD3ϵ PerCP Cy 5.5 (BioLegend, 100218), CD8α Alexa Fluor 700 (BioLegend, 100730), and CD4 APC/Fire 750 (BioLegend, 100460) After surface staining, cells were rinsed with PBS and fixed for 20 min at room temperature using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific,00-5523-00). After fixation cells were stained with antibodies specific for intracellular markers: Granzyme B APC (BioLegend, 372203) and Perforin PE (BioLegend, 154405). Data were collected using the Attune NxT flow cytometer.

Oliveira L, & Drapier J.C. (2000). Down-regulation of iron regulatory protein 1 gene expression by nitric oxide. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6550-6555.

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