Granzyme b ngzb

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Granzyme B (NGZB) is a serine protease enzyme that is primarily expressed in cytotoxic T cells and natural killer cells. It plays a crucial role in the induction of apoptosis, or programmed cell death, in target cells during the immune response.

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Granzyme B (43 citations) Granzyme B (clone NGZB) (3 citations) Anti-Granzyme B (NGZB) (2 citations)

5 protocols using granzyme b ngzb

Multiparameter Flow Cytometry of Tumor Cells

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Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).

Saddawi-Konefka R., Seelige R., Gross E.T., Levy E., Searles S.C., Washington A J.r., Santosa E.K., Liu B., O’Sullivan T.E., Harismendy O, & Bui J.D. (2016). Nrf2 induces IL-17D to mediate tumor and virus surveillance. Cell reports, 16(9), 2348-2358.

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Comprehensive Immune Profiling by Flow Cytometry

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Flow cytometry was performed with Life Attune Nxt FACS machine (Life Technology/Thermo Fisher Scientific, Waltham, MA). Fluoro-chrome conjugated anti-mouse CD45 (30-F11, Biolegend), CD3 (145–2C11, eBioscience), CD4 (RM4-5, eBioscience), CD8 (eBioH35-17.2, eBioscience), Foxp3 (FJK-16s, eBioscience), Ki-67 (SolA15, eBioscience), Granzyme B (NGZB, eBioscience), Ly-6C (AL-21, Biolegend), Ly-6G (1A8, Biolegend), mPD-L1 (10 F.9G2, Biolegend), hPD-L1 (29E.29A3, Biolegend) and IFN-gamma (XMG1.2). Granzyme B and IFN-gamma staining was performed with intracellular staining kit from BD Bioscience. While Ki-67 and Foxp3 staining was done with nuclear protein staining kit from eBioscience (San Diego, CA). FACS data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Huang A., Peng D., Guo H., Ben Y., Zuo X., Wu F., Yang X., Teng F., Li Z., Qian X, & Qin F.X. (2017). A human programmed death-ligand 1-expressing mouse tumor model for evaluating the therapeutic efficacy of anti-human PD-L1 antibodies. Scientific Reports, 7, 42687.

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Tumor Immune Cell Profiling

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The s.c. tumors were minced and then digested in HBSS—10% FBS containing 2 mg/ml collagenase type I (Sigma Aldrich) and 10 μg/ml DNase I (Sigma Aldrich) at 37 °C for 30 min to digestion. Digested cells were passed through a 70 μm pore-size cell strainer to prepare single cell suspensions for flow cytometry. Anti-mouse CD16/32 antibody (93, BioLegend) was pre-added to block the non-specific binding of the immunoglobulin to macrophage Fc receptors. For surface marker analysis, live cells were re-suspended in FACS buffer (1% BSA, 0.1% sodium azide in PBS) and stained with anti-mouse CD45 (30-F11), F4/80 (BM8), Ly6C (HK1.4), CD11b (M1/70, eBioscience), Ly6G (RB6-8C5) (BioLegend), CD206 (MR6F3), CD3 (145-2C11), CD8α (53–6.7), and CD4 (RM4-5) (eBioscience) at 4 °C for 20 min. For intracellular staining, cells were incubated for 2 h with GolgiPlug (BD Biosciences) and GolgiSTOP (BD Biosciences) at 1 μl/ml of culture media. Cells were then surface stained and then fixed and permeabilized using Foxp3/Transcription factor staining Buffer (Invitrogen), labeled with Granzyme B (NGZB, eBioscience) and IFN-γ (XMG1.2, BD Biosciences). Data were acquired using an LSRFortessa system (BD Biosciences) and analyzed with FlowJo software version 10.8.1. (BD Biosciences).

Gu G.J., Chung H., Park J.Y., Yoo R., Im H.J., Choi H., Lee Y.S, & Seok S.H. (2023). Mannosylated-serum albumin nanoparticle imaging to monitor tumor-associated macrophages under anti-PD1 treatment. Journal of Nanobiotechnology, 21, 31.

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Intracellular Cytokine and Cytotoxic Protein Detection

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For intracellular IFN-γ, GzmA, and GzmB detection, splenocytes from control and Ncr1cre Myc^fl/fl mice were left unstimulated or stimulated for a total of 4.5 h at 37°C with 50 ng/ml IL-15 and Brefeldin A (10 μg/ml Enzo Life Science) was added after the first 2 h. The cells were fixed with fixation/permeabilization buffer (eBioscience) for intracellular staining for IFN-γ (F3 IGH48; eBioscience) and granzyme A (3G8.5; eBioscience). Granzyme B (NGZB; eBioscience) staining was performed after fixation (25 min at 22–28°C) and permeabilization with the Foxp3 Transcription Factor Staining Buffer Set (#00-5523-00; eBioscience). Intracellular staining was then performed for 30 min at 22–28°C (with the antibody diluted in the permeabilization buffer).

Khameneh H.J., Fonta N., Zenobi A., Niogret C., Ventura P., Guerra C., Kwee I., Rinaldi A., Pecoraro M., Geiger R., Cavalli A., Bertoni F., Vivier E., Trumpp A, & Guarda G. (2023). Myc controls NK cell development, IL-15-driven expansion, and translational machinery. Life Science Alliance, 6(7), e202302069.

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Flow Cytometric Analysis of Immune Cells

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For flow cytometry, single-cell suspensions of spleen and liver were prepared according to standard protocols. Flow cytometric analysis were performed by using anti-mouse mouse CD8b (53-6.7), CD62L (MEL-14), CD44 (IM7), CD127 (A7R34), KLRG1 (2F1), Fixable Viability Dye eFluor 780, CD45.2 (104), CD45.1 (A20), tumor necrosis factor (TNF) (MP6-XT22), interferon-g (IFN-g) (XMG1.2), IL-2 (JES6-5H4), and granzyme B (NGZB) from eBioscience, preceded by blocking of Fc receptors using 2.4G2 antibodies (in-house generated). To measure cytokine production, cells were stimulated with 10 ng/mL SIINFEKL (N4) peptide for 4 h in the presence of brefeldin A (10 mg/mL; eBioscience). MHC class I tetramers were provided by A. ten Brinke (Amsterdam, the Netherlands). For intracellular staining, permeabilization and fixation of cells was done with the Fix/Perm kit (BD Biosciences). All data were acquired using a FACSVerse (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Kavazović I., Krapić M., Beumer-Chuwonpad A., Polić B., Turk Wensveen T., Lemmermann N.A., van Gisbergen K.P.J.M, & Wensveen F.M. (2022). Hyperglycemia and Not Hyperinsulinemia Mediates Diabetes-Induced Memory CD8 T-Cell Dysfunction. Diabetes, 71(4).

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