Peripheral CD4, CD8, and DP T cells were stimulated with PMA (50 ng/mL) and ionomycin (1.5 μM) and treated with monensin (6.7 μg/mL) for 6 hours at 37°C in a CO2 incubator. After culture, cells were washed with complete medium (RPMI/10% FBS) and resuspended in staining buffer (PBS, 0.5% BSA, and 0.5 mM EDTA). The cells were then fixed, permeabilized, and stained with anti-granzyme B (BioLegend, San Diego, CA, USA) and anti-perforin (Mabtech, Nacka Strand, Sweden) antibodies.
Anti granzyme b
Manufactured by BioLegend
Sourced in United States
Anti-granzyme B is a lab equipment product that detects the presence of granzyme B, a serine protease enzyme involved in the induction of apoptosis in target cells. It is used to measure granzyme B levels in biological samples for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
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Anti cd11c, by BioLegend (3 mentions)
Anti cd3, by BioLegend (3 mentions)
Anti cd45, by BioLegend (3 mentions)
Facsaria 3, by BD (3 mentions)
Anti cd62l, by BioLegend (2 mentions)
Anti perforin, by BioLegend (2 mentions)
Anti tnf α, by BioLegend (2 mentions)
Fixation buffer, by BioLegend (2 mentions)
Intracellular staining permeabilization wash buffer, by BioLegend (2 mentions)
Anti f4 80, by BioLegend (2 mentions)
Anti ifng, by BioLegend (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase d, by Roche (2 mentions)
Fortessa, by BD (2 mentions)
Anti tnf α, by Thermo Fisher Scientific (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
13 protocols using anti granzyme b
1
T Cell Activation and Effector Molecule Expression
2
T Cell Response to Influenza Virus in RIG-I Knockout Mice
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Bone marrow derived dendritic cells (BMDC) were generated from RIG-I+/+ and RIG-I-/- mice and T cell re-stimulation experiments were performed as previously described[31 (link)], [32 (link)]. Briefly, BMDC were first infected with PR8 at an MOI of 0.5 for 5h, washed with PBS 3 times to remove free virus, and resuspended in Iscove's Modified Dulbecco's Media complete media with FBS (IMDM, Invitrogen). For some experiments, infected BMDC were treated with IFN-I (200U/ml) before T cells were added. T cells from naïve or IAV infected RIG-I+/+ and RIG-I-/- mice (day 7 post infection) were enriched using Pan T cells isolation kit II (Miltenyi Biotec) and co-cultured with PR8 infected BMDC at a ratio of 10:1 for 2–3 hours followed by the addition of Brefeldin A (5μg/ml; eBiosciences). DC-T cell co-cultures were further incubated for an additional 8-10hrs. The cells were first surface stained for cell surface markers, followed by intracellular staining for cytokines. For intracellular staining, cells were incubated in permeabilization and fixation buffer (BD Pharmingen) for 45 minutes followed by 2 washes in 1xPBS, 1%FBS, 0.5% Saponin (Sigma, St Louis, MO) and intracellular staining with anti-IFNγ (2μg/ml, XMG1.2) (Biolegend), anti- Granzyme B (2μg/ml, GB11) (Biolegend), anti-TNFα (2μg/ml, MP6-XT22) (eBiosciences) for 30 minutes.
3
Multiparametric Flow Cytometry of Immune Cells
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Different panels of antibody cocktails were generated from anti‐CD45 (BioLegend, 103116, 30‐F11), anti‐CD3 (BioLegend, 100218, 17A2), anti‐CD4 (BioLegend, 100421, GK1x.5), anti‐CD8a (BioLegend, 100711, 53‐6.7), anti‐NKp46 (BioLegend, 137606, 29A1.4), anti‐CD11c (BioLegend, 117307, N418), anti‐CD25 (Biolegend, 101908, 3C7), anti‐FOXP3 (Biolegend, 126404, MF‐14), anti‐granzyme B (BioLegend, 372208, QA16A02), anti‐IFNγ (BioLegend, 505849, XMG1.2), anti‐IFNγ (BioLegend, 505806, XMG1.2, anti‐CD62L (BioLegend, 104432, MEL‐14) and anti‐CD44 (BD Biosciences, 560568, IM7) antibodies, and the AmCyan Live/Dead Cell Staining Kit (Thermo Fisher Scientific). All antibodies were diluted at optimized dilutions before being used.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.
4
Multiparametric Flow Cytometry Analysis of Tumor Immune Microenvironment
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Tumors were digested to single cell suspension enzymatically and filtered twice through 70μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with Cell Staining Buffer (BioLegend), and then stained with the fluorophore-conjugated antibodies for 30 min at 4 °C. The excess of unbound antibodies was washed out before acquisition in flow cytometry. Cells were analyzed using ID700 Spectral Cell Analyzer (SONY) and sorted using FACS-ARIA III (BD, Pharmingen). The following antibodies were for flow cytometry: anti-CD45 PAC (30-F11), anti-CD8b APC-Cy7 (YTS156.7.7), anti-CD4 PE(H129.19), anti-NK1.1 APC-Cy7 (PK136), anti-CD11b APC (M1/70), anti-Ly6c PE (HK1.4), anti F4/80 (BM8), anti-CXCR6 APC (SA051D1), anti-GranzymeB, anti-CD90 FITC (30-H12), anti-CD73 APC (TY/11.8), anti-PD-1 APC (RMP1-30) all purchased from BioLegend and used at 1:100 dilution. Anti-CD31-PE (eBioscience, 390, 1:100), anti-GLUT1 (Novus, Fgi.72, 1:100), anti-Cxcl16 (Bioss, BS-1441R, 1:50) and anti-Rabbit IgG AF488 (Invitrogen, A21245, 1:100) were used to identify CAF populations. Ghost Dye Red 710 (Tonbo, Cat #13-0871-T100) was used to exclude dead cells from the analysis.
5
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.
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.
6
Immune Cell Profiling from Murine Tumors
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Mice were humanely euthanized, and mouse tumors were harvested. Whole tumors were cut and minced into small pieces, followed by digestion in Liberase TL Research Grade 10 (2 µg/mL, Roche, 05401020001) and DNase I (Sigma, 260913-10 MU) in DMEM at 37 °C for 30 min. The digested tissue was filtered through a 70-mm cell strainer (Thermo Fisher Scientific). Cell suspensions were stimulated with PMA (100 µg/mL) plus the protein transport inhibitors Golgi stop and Golgi plug (1:1000, BD Bioscience, 51-2301 KZ) at 37 °C. After 4 h, live cells were identified by vivid yellow staining (Invitrogen, cat# L34959). Cells were stained for cell surface markers including CD45.2 (1:100, BioLegend, 109814), CD8 (1:100, BioLegend, cat# 100708), CD11c (1:100, BioLegend, 117311), and IA/IE (1:100, BioLegend, 107608) at 4 °C for 30 min. For intracellular staining, cells were fixed and permeabilized with a fixation/permeabilization kit (BD Bioscience, 554714) for 30 min at 4 °C and then stained with anti-IFN-γ (1:100, BioLegend, 505813) and anti-granzyme B (1:100, BioLegend, 515406) antibodies. Cells were imaged on a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar).
7
Intracellular Cytokine Staining Optimized
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For intracellular staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA; final concentration 25 ng/mL; Merck, Darmstadt, Germany) and ionomycin (final concentration 1 µg/mL; Merck) for 18 hours. After 2 hours, Brefeldin A (5µg/mL; Merck) and Monensin (1µg/mL; Merck) were added. First, surface antigens were stained as described above. Cells were then permeabilized with fixation/permeabilization solution (Cytofix/Cytoperm; BD Biosciences) and stained with fluorochrome-conjugated antibodies for 30 minutes at 4°C. The following anti-human monoclonal antibodies were used: anti-IL-2, anti-CD4, anti-IL-10, anti-TCR-γ/δ (BD Biosciences), anti-TCR-Vδ2 (Beckman Coulter Life Sciences), anti–IFN-γ, anti-TNF-α, anti-CD8, anti-TGF-β, anti-CD39, anti-Granzyme-B, anti-CD19, anti-CD3, anti-CD73, and anti-CD14 (all BioLegend), see also Supplementary Table 3
For kinetic studies of CD39 surface expression, cells were stimulated as previously described with small adaptations (109 (link)). Briefly, cryopreserved PBMC were plated into 48-well plates and stimulated with rhIL-2 (20 U/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), PMA (5 ng/mL), ionomycin (0,5 µg/mL), anti-CD3/CD28-Dynabeads (ratio 1:1; ThermoFisher Scientific, Waltham, USA) or combinations thereof. Cells were cultured for up to 6 days before FACS analysis.
For kinetic studies of CD39 surface expression, cells were stimulated as previously described with small adaptations (109 (link)). Briefly, cryopreserved PBMC were plated into 48-well plates and stimulated with rhIL-2 (20 U/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), PMA (5 ng/mL), ionomycin (0,5 µg/mL), anti-CD3/CD28-Dynabeads (ratio 1:1; ThermoFisher Scientific, Waltham, USA) or combinations thereof. Cells were cultured for up to 6 days before FACS analysis.
8
Multi-Dimensional Immune Cell Analysis
9
Tumor Infiltrating Immune Cell Analysis
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Briefly, 4 different group tumor tissues were digested at 37 °C for 30 minutes with 1 mg/mL Collagenase D and 0.1 mg/mL DNase I (Roche). Digestion was stopped by EDTA, and cells were filtrated through 70-mm cell strainers and washed twice with PBS containing 1 mM EDTA and 2% fetal bovine serum (FBS) (staining buffer). Cells were re-suspended in the staining buffer and stained with following antibodies on ice for 30 minutes: anti-CD45, anti-CD8, anti-IFNg, anti-Granzyme B, anti-CD11b, anti-CD11c, anti-FOXP3, anti-CD25, anti-F4/80, Ly6C were purchased from BioLegend. For intracellular staining, cells were fixed with fixation buffer (Biolegend) on ice for 15 minutes, and then washed twice with Intracellular Staining Permeabilization Wash Buffer (Biolegend). Antibodies against IFN-g (Clone XMG1.2) and Granzyme B (Clone: QA16A02) were added and incubated for 1 hour on ice. The cytokine producing cells were determined by flow cytometry. The flow cytometry data were collected on Fortessa (BD) and analyzed by FlowJo (Tree Star). For cell sorting, CD8+ T cells that were co-cultured with tumor cells for 6 hours were collected and washed with culture medium. Re-suspended cells were stained with anti-CD8a antibodies (Clone: 53-6.7) for 30 min on ice. After a washing step, cells were sorted on a BD FACS AriaIII (BD) and lysed in the buffer RLT plus (QIAGEN).
10
Immune Profiling of Tumor Cell Lines
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Cell suspensions from KPC-Brca2–/– shCtrl and shPOLQ tumors were prepared using the Mouse Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s recommendations. Single cells from tumor digestion were incubated with 2 μg/mL anti-mouse CD3 (BD Biosciences, 553057), 1 μg/mL anti-mouse CD28 (BD Biosciences, 553294), and 1 μL/mL Cell Stimulation Cocktail (eBioscience) for 4 hours at 37°C in RPMI with 10% FBS. Cells were stained extracellularly with specific antibodies against mouse CD3 (557596), CD4 (560782), CD8 (563046), CD45 (560510), CD11c (563048), CD11b (562127), CD69 (553237), Ly6C (561237), F4/80 (565853), Ly6G (130-119-902), all from Miltenyi Biotec, and CK19 (ab192643), from Abcam. After washing, cells were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience) and stained intracellularly with anti-IFN-γ (BD Biosciences, 561040) and anti-granzyme B (BioLegend, 515405). Samples were acquired on a flow cytometry analyzer (Attune NxT; Thermo Fisher Scientific). Data were analyzed with FlowJo software (BD Biosciences).
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