Anti perforin

Manufactured by BioLegend

Sourced in United States

Anti-Perforin is a monoclonal antibody that binds to the perforin protein. Perforin is a cytolytic protein found in the cytoplasmic granules of cytotoxic T cells and natural killer cells and is involved in cell-mediated immunity. The Anti-Perforin antibody can be used for the detection and quantification of perforin expression.

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Anti-Perforin-PE (9 citations) Anti-perforin-APC (6 citations) APC anti-human Perforin (3 citations) Anti-human perforin (clone dG9; (2 citations) PE-anti-perforin (clone B-D48, (2 citations) Anti-mouse Perforin Antibody(S16009A), (2 citations) Anti-perforin clone dG9 (2 citations) Anti-Perforin-FITC (2 citations) APC anti-mouse Perforin (2 citations) Anti-human Perforin (2 citations)

7 protocols using anti perforin

Assessing T-cell Responses to Pancreatic Cancer Cells

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CD4+ and CD8+ T-cells were magnetically sorted (CD4 Microbeads Human; #130-045-101 and CD8 Microbeads Human; #130-045-201; Miltenyi Biotech), stained with 5 µM CFSE (#V12883; Invitrogen, Carlsbad, CA, USA) and incubated with mitomycin c-treated PDAC cell lines in U-bottom 96 well plates for 4 days. Allogeneic mature DCs were used as positive controls. Where indicated, purified endotoxin-free anti-MHC class I (clone W6/32; #311428; BioLegend), anti-MHC class II (clone TÜ39; #555556; BD Bioscience, Chicago, IL, USA), anti-LAG-3 (clone 17B4; Novus Biologicals; #NBP1-97657), anti-PD-L1 (clone 29E.2A3; #329716; BioLegend) or a combination of anti-LAG-3 and anti-PD-L1 antibodies were added at 10 μg/mL. The results are expressed as proliferation index, determined by dividing the mean fluorescence intensity (MFI) of CFSE in T-cells alone by the MFI of the T-cells in the co-cultures. Supernatants were collected for the assessment of IFN-γ and granzyme B by ELISA. Cells were stained with fluorochrome-labeled anti-CD4 (#317422; BioLegend), anti-CD8 (#300918; BioLegend) and anti-perforin (#353305; BioLegend) and processed for flow cytometry.

Baleeiro R.B., Bouwens C.J., Liu P., Di Gioia C., Dunmall L.S., Nagano A., Gangeswaran R., Chelala C., Kocher H.M., Lemoine N.R, & Wang Y. (2022). MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy. Oncoimmunology, 11(1), 2080329.

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Comprehensive NK Cell Phenotyping

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The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.

Chen M., Li Y., Wu Y., Xie S., Ma J., Yue J., Lv R., Tian Z., Fang F, & Xiao W. (2021). Anti-Tumor Activity of Expanded PBMC-Derived NK Cells by Feeder-Free Protocol in Ovarian Cancer. Cancers, 13(22), 5866.

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Multi-Dimensional Immune Cell Analysis

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Single cell suspensions of splenocytes or lymph nodes were surface-stained with anti-NK1.1 and CD3. Then the cells were fixed, permeabilized and stained with anti-granzyme B, anti-perforin or anti-IFNγ (BioLegend). Data were acquired by BD LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star). Follicular helper T cells (T_FH) and T_reg cells in splenocytes or lymph nodes were identified as CD4⁺CD62L⁻CD44⁺CXCR5⁺Bcl6⁺ and CD4⁺CD62L⁻CD44⁺Foxp3⁺ cells (antibodies all from eBioscience otherwise noted) respectively CXCR5 is stained by 3 steps: first, the cells were incubated with purified anti-CXCR5, followed by biotinylated goat anti-rat IgG (Jackson ImmunoResearch) then by streptavidin conjugated with PE-Cy7 (eBioscience).

Kawakami Y., Ando T., Lee J.R., Kim G., Kawakami Y., Nakasaki T., Nakasaki M., Matsumoto K., Choi Y.S, & Kawakami T. (2016). Defective NK cell activity in a mouse model of eczema herpeticum. The Journal of allergy and clinical immunology, 139(3), 997-1006.e10.

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Comprehensive Immune Cell Profiling in Tumor Samples

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Fresh tumor and spleen samples were minced, treated with 200 U/ml Collagenase I (Cat#ST2294, Beyotime Biotechnology), and filtered with a 100 μm filter to make a suspension of single cells. 2 × 10^{6 (link)} Cells were stained at 100 μl in PBS. Single-cell suspension is stained with the following antibodies: Live/Dead Fixable Violet Dead Cell (Cat# 2549272, Invitrogen), anti-CD3 (Cat# 100236, Biolegend), anti-CD4 (Cat# 100406; 100408, Biolegend), anti-CD8 (Cat# 100706, Biolegend), anti-Ki67 (Cat# 652425, Biolegend), anti-IFN-γ (Cat# 505860, Biolegend), anti-Perforin (Cat# 154306, Biolegend), anti-Foxp3 (Cat# 126404, Biolegend), anti-CD11b (Cat# 101212, Biolegend), anti-CD11c (Cat# 117352, Biolegend), anti-Gr-1(Cat# 108406, Biolegend), anti-TIM-3(Cat# 119727, Biolegend), anti-LAG-3 (Cat# 125241, Biolegend) and anti-PD-1 (Cat# 135227, Biolegend). Surface antigens were detected by NovoCyte Quanteon (Agilent) after staining with antibodies for 30 minutes in the dark. To detect intracellular cytokines, the samples were fixed with Fixation/Permeabilization Solution KiT (Cat# 554714, BD Biosciences) and incubated with intracellular antibodies for 30 minutes before running the machine. Datas were analyzed by NovoExpress.

Wei W., Tian L., Zheng X., Zhong L., Chen Y., Dong H., Zhang G., Wang S, & Tong X. (2024). Expression of GPX4 by oncolytic vaccinia virus can significantly enhance CD8+T cell function and its impact against pancreatic ductal adenocarcinoma. Oncoimmunology, 13(1), 2322173.

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Comprehensive Cell Apoptosis Assay

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For cell apoptosis assay, tumor cells were collected after citrate treatment and detected with PE‐Annexin V Apoptosis Detection Kit (BD Biosciences). The markers on tumor cells or immune cells were examined by the flow cytometry analysis after surface staining or intracellular staining indicated antibodies. The antibodies for DNA damage markers included: anti‐phospho‐ATM (Ser1981, #13 050), anti‐phospho‐H2AX (Ser139/Tyr142, #5438), anti‐phospho‐CHK2 (Thr68, #2661), and anti‐phospho‐53BP (Ser25/29, #2675) (1:200 dilution, Cell Signaling Technology). The antibodies for immune cells included: anti‐CD3 (17A2), anti‐CD4 (GK1.5), anti‐CD8 (53–6.7), anti‐CD19 (6D5), anti‐NK1.1 (PK136), anti‐IFN‐γ (XMG1.2), anti‐IL‐4 (11B11, BD), anti‐IL‐10 (JES5‐16E3), anti‐IL‐17A (TC11‐18H10.1), anti‐granzyme B (GB11), and anti‐perforin (S16009A), which were all purchased from Biolegend. All stained cells were analyzed on a Guava EasyCyte Plus Flow Cytometer (Millipore) and data were analyzed with the FlowJo software (Tree Star).

Zhao Y., Liu X., Si F., Huang L., Gao A., Lin W., Hoft D.F., Shao Q, & Peng G. (2021). Citrate Promotes Excessive Lipid Biosynthesis and Senescence in Tumor Cells for Tumor Therapy. Advanced Science, 9(1), 2101553.

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Multiparametric Immune Profiling of PBMCs

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After PBMCs stimulation, cells were washed with PBS and incubated at 4°C for 30 minutes with Fixable Viability Dye eFluor 506 (eBioscience), and with the anti-CD3-AF700 (clone: UCHT1, eBioscience) and anti-CD8-eFluor450 (clone: RPA-T8, eBiocience) antibodies. Next, cells were fixed and permeabilized with Foxp3 Fixation/Permeabilization Buffer (eBioscience) and then incubated with the following conjugated antibodies: anti-IL-2- (clone: 5344.111, BD), anti-Granzyme B (clone: GB11, BD), anti-Perforin (clone: B-D48, Biolegend), anti-IFN-γ (clone: 4S.B3, Biolegend), anti-TNF-α (clone: MAb11, eBioscience) and anti-IL-10 (clone: JES3-9D3, eBioscience). Cells were acquired on an LSR Fortessa flow cytometer using the BD FACSDiva software v 8.0.1 (BD). At least 100,000 CD3⁺ events were recorded. Fluorescence minus one (FMO) control for the effector molecules was included to define positive thresholds. For fluorochrome spillover compensation, unstained and single-stained cells were used with each of the fluorochrome-labeled antibodies. Then, automatic compensation was performed on LSR Fortessa.

Sánchez-Martínez A., Acevedo-Sáenz L., Alzate-Ángel J.C., Álvarez C.M., Guzmán F., Roman T., Urcuqui-Inchima S., Cardona-Maya W.D, & Velilla P.A. (2022). Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia. Frontiers in Immunology, 13, 793982.

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Characterizing Immune Cells by Flow Cytometry

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TILs and PBLs were suspended in flow buffer (PBS containing 2% fetal bovine serum), and incubated with anti-CD3 and anti-CD8 (Biolegend, San Diego, CA, USA) against surface antigens for 30 min at 4 °C in the dark, with a subsequent wash in flow buffer, spin and resuspension. Cells were then fixed, permeabilized and stained with anti-IFN-γ, anti-Perforin and anti-Granzyme B antibody (Biolegend, San Diego, CA, USA) for 30 min at 4 °C in the dark at each step. After staining, cells were analyzed using a BD CantoII flow cytometer (Becton Dickinson, San Jose, CA, USA). To detect nonspecific signals, concentration- and isotype-matched nonspecific antibodies were used.

Gao Q., Wang S., Chen X., Cheng S., Zhang Z., Li F., Huang L., Yang Y., Zhou B., Yue D., Wang D., Cao L., Maimela N.R., Zhang B., Yu J., Wang L, & Zhang Y. (2019). Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC. Journal for Immunotherapy of Cancer, 7, 42.

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