Granzyme b fitc

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Granzyme B-FITC is a fluorescently-labeled antibody that binds to and detects the presence of Granzyme B, a serine protease enzyme involved in cell-mediated cytotoxicity. This product can be used in various immunoassays and flow cytometry applications to quantify and analyze Granzyme B expression.

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Granzyme B (61 citations) Anti-Granzyme B-FITC (6 citations) FITC anti-human/mouse Granzyme B (5 citations) FITC anti-human/mouse Granzyme B Antibody (4 citations) Granzyme B FITC (clone GB11, (3 citations) Human/mouse granzyme B–FITC (2 citations) FITC-conjugated anti-granzyme B (clone GB11), (2 citations) FITC-Granzyme B- clone (2 citations) FITC anti-human granzyme B (2 citations) FITC-Granzyme B antibody (2 citations)

9 protocols using granzyme b fitc

Multicolor Flow Cytometry Panel

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FITC-Granzyme B (515403), FITC-Ly6C (128006), PE-PD-L1 (124308), BV421-F4/80 (123124), APC-NK1.1 (108710), APC-CD11c (117310), APC-CD44 (103012), APC-Cy7-CD19 (115530), APC-Cy7-I-A/I-E (107628), PE-Cy7-CD8a (100722), PE-Cy7-CD206 (141720), BV605-CD4 (100548), BV605-CD11b (101237), BV711-CD69 (104537), BV711-CD45R/B220 (103255), BV711-Tbet (644819), Percp-Cy5.5-CD45 (147706) were purchased from BioLegend (San Diego, California). PE-CD62L (12-0621-82), PE-Foxp3 (12-5773-82), Pacific Blue-Ki67 (48-5698-82), APC-eFluor780-PD1 (47-9985-80), PE-Cy5-CD3 (15-0031-81), PE-Cy5-Eomes (15-4875-80), anti-CD16/32 (14-0161-85) were purchased from Thermo Fisher (Waltham, Massachusetts).

Xin B., Yang M., Wu P., Du L., Deng X., Hui E, & Feng G.S. (2022). Enhancing the Therapeutic Efficacy of PD-L1 Antibody for Metastasized Liver Cancer by Overcoming Hepatic Immunotolerance. Hepatology (Baltimore, Md.), 76(3), 630-645.

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Isolation and Characterization of Tumor-infiltrating NK Cells

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NK cells were harvested and isolated from spleens of tumor bearing mice. Processed into single cell suspension following above protocol, and NK were isolated using an NK isolation kit (Stemcell) following manufacturer’s instructions. Isolated NKs were then stained using the following antibodies: APC-EpCAM, PE/Dazzle 594- NKp46 (Biolegend), FITC-Granzyme B (Biolegend). Nuclei were stained using 4′6-diamidino-2-phenylindole (DAPI). Following staining, cells were washed and resuspended in PBS and processed through Amnis Imagestream X Mk II imaging flow cytometer (Amnis, Seattle, WA). Analysis was performed using IDEAS 6.2 software (AMNIS, Seattle, WA).

Gadwa J., Amann M., Bickett T.E., Knitz M.W., Darragh L.B., Piper M., Van Court B., Bukkapatnam S., Pham T.T., Wang X.J., Saviola A.J., Deak L.C., Umaña P., Klein C., D’Alessandro A, & Karam S.D. (2023). Selective targeting of IL2Rβγ combined with radiotherapy triggers CD8- and NK-mediated immunity, abrogating metastasis in HNSCC. Cell Reports Medicine, 4(8), 101150.

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Single Cell Profiling of Tumor-Infiltrating T Cells

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Single-cell suspensions were prepared and resuspended in a staining buffer (BD Biosciences, #554656). For the in vitro EphA10 antibodies binding assay, 1 × 10⁶ BT-549 EphA10 overexpressing tumor cells were stained with 0.2 μg of anti-EphA10 #4, #8, #9, and IgG1 isotype control (Bio X cells, #BE0083) in 100 μl staining buffer for 30 min at RT. After the cells were washed by staining buffer three times, they were stained with APC anti-mouse second antibody according to a standard protocol. To analyze CTL profiles in mouse tumor samples, a Mouse Tumor Dissociation Kit (Miltenyi Biotec, #130-096-730) and gentle MACS Octo Dissociator (Miltenyl Biotec, #130-096-427) were used to digest mouse tumors into single cells. After red blood cells were removed and hybridized with TruStain FcX CD16/CD32 antibody (1:50; BioLegend, #101319), single cells were stained for flow cytometry according to standard protocols with antibodies against the following: PE-CD45 (1:200; BioLegend, #103105), APC-CD3ε (1:100; BioLegend, #100311), APC/Cy7-CD8a (1:100; BioLegend, #100713), and PreCP-CD4 (1:100; BioLegend, #100537). For further intracellular staining, cells were fixed, permeabilized, and stained with FITC-Granzyme B (1:50; BioLegend, #515403). Stained cells were analyzed with use of a BD FACSCanto II cytometer (BD Biosciences). Data were processed by using the FlowJo software program.

Cha J.H., Chan L.C., Wang Y.N., Chu Y.Y., Wang C.H., Lee H.H., Xia W., Shyu W.C., Liu S.P., Yao J., Chang C.W., Cheng F.R., Liu J., Lim S.O., Hsu J.L., Yang W.H., Hortobagyi G.N., Lin C., Yang L., Yu D., Jeng L.B, & Hung M.C. (2022). Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer. The Journal of Biological Chemistry, 298(4), 101817.

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Characterization of NK Cell Activation

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To assess proliferation and further characterisation of the differentiation of NK cells, PBMC were permeabilised and stained with anti-Ki67-PE (eBioscience, Hatfield, UK), Granzyme-B-FITC and Perforin-Cy5.5/PerCP (Biolegend, London, UK) directly ex-vivo. For intracellular staining for IFNγ production; PBMC were incubated with rhIL12 and rhIL15 (10ng/ml) (R&D systems, Abingdon, UK), for 19 hours at 37°C. 1mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 hours. Cells were then stained with anti-CD3-Cy5.5/PerCP or CD3/Pe-Cy7, CD16-APCy7, CD56-FITC, and subsequently fixed and permeabilised, followed by intracellular staining for IFNγ-v450 (BD Biosciences, Oxford, UK). Dead cells were excluded by fixable live dead stain.
For degranulation, PBMCs were incubated with K562 cells (5:1 E:T ratio) for 3 hours following overnight stimulation with a combination of 50ng/ml rhIL12 and rhIL18 (Miltenyi Biotech). Anti-CD107a-PE mAb (BD Biociences, Oxford, UK) was added at the time of stimulation with target-cells and monensin (1mM) added during the last 2 hours of incubation prior to staining and acquisition.

Gill U.S., Peppa D., Micco L., Singh H.D., Carey I., Foster G.R., Maini M.K, & Kennedy P.T. (2016). Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo. PLoS Pathogens, 12(8), e1005788.

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Differential Immunophenotyping of NK Cell Subsets

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The following antibodies were used. From eBioscience (San Diego, CA): CD3-APC eFluor 780 (clone SK7); CD16-FITC (eBioCB16); CD19-APC eFluor 780 (HIB19); CD45-PE (HI30); CD94-FITC (DX22); Eomes-PE eFluor 610 (WD1928); Granzyme K-PerCP eFluor 710 (G3H69); HLA-A3-FITC (GAP.A3); IFN gamma-Alexa Fluor 488 (4S.B3); S1PR1-eFluor 660 (SW4GYPP); T-bet-PECy7 (4B10); TNF alpha-APC (Mab11). From Biolegend (London, UK): CCR5-APC (J418F1); CD49a-FITC (TS2/7); CD69-APC (FN50); CD103-FITC (Ber-ACT8); CX3CR1-FITC (2A9-1); CXCR6-PerCP Cy5.5 (K041E5); CXCR6-APC (K041E5); GM-CSF-PE (BDV-21C11); Granzyme B-FITC (GB11); HLA-A2-FITC (BB7.2); KIR2DL1/S1/S3/S5-APC (HP-MA4); KIR2DL2/L3-APC (DX27); KIR3DL1-APC (DX9); Perforin-APC (dG9). From BD (Oxford, UK): CD56-BV510 (NCAM16.2), LIF-PE (1F10). Dead cells were excluded using Fixable Viability Dye eFluor 450 (eBioscience). Intracellular staining was carried out using Human FoxP3 Buffer (BD) according to the manufacturer’s instructions. Data were acquired on a Fortessa II (BD) and analysed using FlowJo (Treestar, Ashland, OR). Cells were sorted on an Aria (BD). Eomes^lo NK cells were isolated by sorting on live cells (propidium iodide negative, Tonbo Biosciences, San Diego, CA), singlets, scatter, CD3^- CD56⁺ CXCR6^- CD16⁺. Eomes^hi NK cells were isolated by sorting on live cells, singlets, scatter, CD3^- CD56⁺ CXCR6⁺.

Cuff A.O., Robertson F.P., Stegmann K.A., Pallett L.J., Maini M.K., Davidson B.R, & Male V. (2016). Eomeshi NK Cells in Human Liver Are Long-Lived and Do Not Recirculate but Can Be Replenished from the Circulation. The Journal of Immunology Author Choice, 197(11), 4283-4291.

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Characterization of Primary Human NK Cells

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NK cell markers expression was verified using mouse anti-human antibodies to CD45-APC-CY7, CD25-FITC, CD16-PE, CD3-PE-CY7, CD56-PAC BLUE and CD122-PE, were from BD Biosciences. Anti-human antibodies to NKG2D-APC, MHC-1 HLA-A2-APC, NKP30-PE, NKP44-PE-CY7, NKP46-FITC, Granzyme B-FITC, Perforin1-PE, Interferon-γ-APC, TNF-α1- APC-CY7, were from BioLegend, DAPI from Invitrogen, mouse anti-cMyc: sureLight APC was from Columbia Biosciences. Cells were sorted at MGH Flow Cytometry Core facility using a BD 5 laser SORP FACS Vantage SE Diva system (BD Biosciences). FACS data and ∑Median statistics were analyzed using FlowJo Software (Tree Star, Inc.). Human primary NK (hNK) cells were extracted from peripheral blood of healthy donors using the Rosettesep™ human enrichment kit (StemCell technologies).

Jounaidi Y., Cotten J.F., Miller K.W, & Forman S.A. (2017). Tethering IL2 to its receptor IL2Rβ enhances anti-tumor activity and expansion of natural killer NK92 cells. Cancer research, 77(21), 5938-5951.

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Immunophenotyping of Human Lymphocyte Subsets

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The mAbs used to stain human immune cells included PD-1-APC (Clone: EH12.2H7, eBioscience, San Diego, CA), CTLA-4-PE (Clone: L3D10), TIM-3-Brillian violet 421 (Clone: F38-2E2), Granzyme B-FITC (Clone: GB11), Perforin-APC (Clone: dG9, Biolegend, San Diego, CA), CD3-Alexa Fluor 405 conjugate (Clone: UCHT1, Invitrogen, Grand Island, NY) and CD4-AF700 (Clone RPA-T4, BD Biosciences, San Diego, CA) and their respective isotype controls. All mAb preparations were pre-titrated using activated as well as non-activated PBMC to determine the optimal staining dilution for each. Intracellular staining for granzyme B, and perforin was performed as follows: PBMCs or TILs were stained with mAb for surface markers and subsequently fixed and permeabilized (eBioscience). After washing, cells were subjected to intracellular staining for Foxp3, granzyme B, and perforin. Flow cytometry was performed using an EPICS XL-MCL flow cytometer equipped with Expo32 software, a CyAn flow cytometer (Dako, Ft. Collins, CO) or Fortessa (Becton Dickinson) machine; data were analyzed using Summit V4.3 software or the flowJo software (TreeStar, Inc.). The acquisition and analysis gates were restricted to the lymphocyte gate based on characteristic properties of the cells in the forward and side scatter. At least 1 × 10⁵ events were acquired for analysis.

Jie H.B., Srivastava R.M., Argiris A., Bauman J.E., Kane L.P, & Ferris R.L. (2017). Increased PD-1+ and TIM-3+ TILs during cetuximab therapy inversely correlates with response in head and neck cancer patients. Cancer immunology research, 5(5), 408-416.

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Multiparametric Flow Cytometry Analysis

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Thymi, spleens, tumors and lungs were collected, and single-cell suspensions were made. The cells were then treated with RBC lysis buffer, washed, and blocked in PBS supplemented with Fc Blocker (BioLegend #422302) for 15 min and then stained with antibodies against surface makers for 30 min. After staining, cells were washed two times in PBS without Mg⁺⁺ or Ca²⁺ and then fixed in 4% PFA (paraformaldehyde) before analysis on a BD Fortessa. For intracellular staining, after fixation, cells were permeabilized in 0.1% Triton X-100 followed by staining with intracellular markers. Most of the flow cytometry antibodies were purchased from BioLegend (CD45-PerCP#103130, CD3-BV650#100229, CD4-BV421#100443, CD8-PerCP#100732, CD8-PE#140408, IFNγ-APC#505809, Granzyme B-FITC#515403, Siglec-E-APC#677105). Live/dead fixable stain Aqua #L34957 and Green #L23105 were obtained from Thermo Fisher Scientific. LIN28A-AF488 antibodies were from Cell Signaling Technology (#12573S).

Carter J.A., Matta B., Battaglia J., Somerville C., Harris B.D., LaPan M., Atwal G.S, & Barnes B.J. (2023). Identification of pan-cancer/testis genes and validation of therapeutic targeting in triple-negative breast cancer: Lin28a-based and Siglece-based vaccination induces antitumor immunity and inhibits metastasis. Journal for Immunotherapy of Cancer, 11(12), e007935.

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Multiparameter Flow Cytometry Analysis

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Single cells were incubated with Human TruStain FcX (BioLegend) to block Fc receptors. These were then stained with Ghost Dye™ Red 780 Viability Dye (Cell Signaling Technology) to identify dead cells and the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD103-PerCP/Cy5.5 (Ber-ACT8), CTLA-4- PerCP/Cy5.5 (BNI3), Granzyme B-FITC (QA16A02), PD-1-PE (EH12.2H7), CD27- PE/Cy7 (LG.3A10), CD3- PE/Cy7 (HIT3a), CD8a-AF700 (RPA-T8), CD4-APC (OKT4), CD127-BV711 (A019D5), Ki-67-BV605 (Ki-67), CD45RA-BV605 (HI100), CCR7-BV510 (G043H7), CD69-BV421 (FN50), and IFNγ-BV421 (4S.B3) (all from BioLegend). Data were acquired on a LSRFortessa X-20 (BD) and analyzed with FlowJo software.

Miyabe C., Dong Y., Ikeda T., Takahashi K., Miyabe Y, & Kawakami T. (2021). Immune checkpoint molecule expression is altered in the skin and peripheral blood in vasculitis. Scientific Reports, 11, 20019.

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