> Chemicals & Drugs > Amino Acid > Granzymes

Granzymes

Granzymes are a family of serine proteases found in the cytotoxic granules of natural killer cells and cytotoxic T lymphocytes.
They play a key role in the induction of apoptosis in target cells during the cell-mediated immune response.
Granzymes have been implicated in a variety of physiological and pathological processes, including tumor suppression, autoimmune disorders, and inflammation.
Understanding the mechanisms of granzyme function is crucial for developing effective therapies targeting these important immune effector moleccules.

Most cited protocols related to «Granzymes»

Immunohistochemical Analysis of COVID-19 Skin Lesions

Cited 12 times

Samples of COVID‐19‐associated perniosis were sectioned at a thickness of 5 µm and stained on positively charged glass slides stored at 4 °C within 3 days after sectioning. Deparaffinization, rehydration and antigen retrieval were performed on the BOND‐III Leica automated slide stainer (Leica Biosystems, Newcastle upon Tyne, UK) following the methodologies that have been previously described.¹⁴ (link), ¹⁵ (link) Specimens were incubated and variably stained with CD3 (LN10), CD4 (4B12), CD8 (4B11), CD7 (LP15), CD11c (5D11), CD20, myeloperoxidase, and granzyme (11F1) from Leica Biosystems; lysozyme (Rabbit Poly) from Agilent (Santa Clara, CA, USA); and myxovirus virus resistance protein A (MXA, C‐1) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) at room temperature, followed by visualization with the Leica BOND detection kit for 20 min at room temperature.¹⁶ (link) The specimens were then counterstained with haematoxylin and coverslipped.¹⁴ (link), ¹⁵ (link) For the cases of thrombotic retiform purpura, immunohistochemical staining of C3d (ready‐to‐use polyclonal antibody; Cell Marque, Rocklin, CA, USA), C4d (1: 50, monoclonal antibody; Quidel, San Diego, CA, USA), mannan‐binding lectin serine peptidase (MASP)‐2 (Sigma, St Louis, MO, USA; HPA029313) and C5b‐9 (1: 250, clone aE11; Dako, Glostrup, Denmark) was accomplished using the BOND‐III autostainer.
Further viral studies were performed on the cases of COVID‐19‐associated perniosis and COVID‐19 thrombotic retiform purpura and three cases of perniosis unrelated to COVID‐19. Positive and negative controls were used with every run, including autopsy lung tissue from patients with COVID‐19 pneumonia and normal lung tissue obtained prior to 2019, respectively. Antibodies specific for the envelope protein (1: 300) and spike protein (1: 7000) of COVID‐19 (ProSci, Poway, CA, USA) were optimized with the lung tissues. Optimal pretreatment conditions included ethylenediaminetetraacetic acid antigen retrieval solution (pH 9·0) for 30 min. The analyses were done on the automated Leica BOND platform, with the modification that the Enzo Life Sciences (Farmingdale, CA, USA) peroxidase antimouse/rabbit conjugate (catalogue #ADI‐950‐113‐0100) was used in place of the equivalent Leica conjugate as this reduced background.
Detection of SARS‐CoV‐2 RNA was done using the ACD RNAscope probe (catalogue #848561‐C3; ACD, Newark, CA, USA) as per a previously published protocol in which only the viral RNA probe is changed. The SARS‐CoV‐2 probe targets the target region nt21631–23303 derived from the genomic sequence of the virus (NC_045512·2). In brief, pretreatment in the ACD RNA retrieval solution and protease digestion is followed by overnight hybridization at 37 °C and detection using the ACD 2·5 High Definition diaminobenzidine detection kit.

Magro C.M., Mulvey J.J., Laurence J., Sanders S., Crowson A.N., Grossman M., Harp J, & Nuovo G. (2021). The differing pathophysiologies that underlie COVID‐19‐associated perniosis and thrombotic retiform purpura: a case series. The British Journal of Dermatology, 184(1), 141-150.

Full text: Click here

Publication 2021

acid citrate dextrose Antibodies Antigens Autopsy Cells Chilblains Clone Cells Complement Membrane Attack Complex COVID 19 Crossbreeding Digestion Edetic Acid Gene Products, env Granzymes Hematoxylin Immunoglobulins Lung Mannose-Binding Protein-Associated Serine Proteases Mannose Binding Lectin Monoclonal Antibodies M protein, multiple myeloma Muramidase Myxoviruses Patients Peptide Hydrolases Peroxidase Pneumonia Poly A Purpura Rabbits Rehydration RNA Probes SARS-CoV-2 Serine Proteases Tissues Viral Genome Viral Proteins

Single-sample gene set enrichment analysis

Cited 6 times

Single-sample gene set enrichment analysis (ssGSEA) was performed to calculate gene set enrichment scores for each sample ^{21 (link)} using the ssGSEAProjection module from the GenePattern server (https://genepattern.broadinstitute.org/gp) ^{22 (link)}. The MYCN-signature gene set consists of 157 genes up- or down-regulated by MYCN ^{23 (link)}. The immune score was calculated using the ESTIMATE method ^{24 (link)}, and 22 immune cells-specific gene sets were derived from a published study ^{25 (link)}. We also used a cytolytic signature, an average expression of six genes, 5 granzymes (GZMA, GZMB, GZMH, GZMK, GZMM) and perforin (PRF1), similar to what was described by Rooney et al^{26 (link)}, to quantify the level of cytotoxic immune cell activity. Genes that did not have approved HUGO Gene Nomenclature Committee (HGNC) symbols were excluded from the ssGSEA analysis. The ssGSEA scores for TARGET and the validation samples were calculated using a subset of genes shared by both datasets to facilitate the direct comparison of the scores across the datasets (Supplementary Table S3).

Wei J.S., Kuznetsov I.B., Zhang S., Song Y.K., Asgharzadeh S., Sindiri S., Wen X., Patidar R., Najaraj S., Walton A., Auvil J.M., Gerhard D.S., Yuksel A., Catchpoole D., Hewitt S.M., Sondel P.M., Seeger R., Maris J.M, & Khan J. (2018). Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T Cell Receptors in High-risk MYCN-not-amplified Human Neuroblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(22), 5673-5684.

Publication 2018

Gene Expression Genes Genes, vif Granzymes GZMA protein, human GZMB protein, human GZMM protein, human Lanugo MYCN protein, human Perforin

Comprehensive T Cell Activation Assays

Cited 4 times

T cell activation assays were performed using primary CD31⁺ CD8T cells purified from PBMC or SF (negative selection with Rosette Sep, Stem Cell Tech). Jurkat and JRT3 cells (ATCC) were used as internal system controls. Activation bioassays with 2 μg/ml anti-TCRαβ (1P26, Biolegend) or 5 μg/ml anti-CD3 (OKT3, Orthoclone) followed established procedures (15 (link), 20 (link)). For stimulations using anti-CD31 (WM59, eBioscience) and recombinant CD38-Ig (R&D Systems), optimal concentrations of 1 and 10 μg/ml, respectively, were empirically determined. Stimulating agents were either cross-linked in solution with species-specific anti-Ig, or immobilized in tissue culture plates following published procedures (15 (link), 20 (link)). All activation assays included Ig isotype and media only controls.
At the indicated time periods, cells were harvested and immunostained for CD25, CD69, phospho-tyrosine (4G10, Millipore), and granzyme/CD107a (BD). The latter two molecules were determined from permeabilized cells using Cytoperm reagent (BD). For granzyme detection, Golgi Plug/Stop reagent (BD) was added to cultures during the last 6 hours of incubation. Markers of cell activation were measured by multicolor flow cytometry as described above. The culture supernatants were also harvested, and the cytokine content measured by the Luminex system (6 (link), 15 (link)).
Proliferation assays on primary T cells in PBMC or SF were performed using standard bioassays using 5- (& 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (6 (link)). Subsets of dividing or non-dividing cells were identified by immunostaining for CD4, CD8, CD28, CD31, and γH2AX after 5 days in culture.
For the analysis of in vitro senescence cultures, purified CD28⁺ T cells from PBMC were subjected to repeated stimulations with irradiated allogeneic PBMC and EBV-transformed lymphoblastoid cells, anti-CD3 (OKT3), and recombinant interleukin (IL)-2 (Proleukin, Chiron) as described previously (21 (link)). Cell phenotypes of the cultures were determined by flow cytometry at 7 days after each stimulation cycle.
For T cell population doubling analysis, cultures of T cells from PBMC or SF were monitored according to the procedure of Koetz et al (22 (link)).

Dvergsten J.A., Mueller R.G., Griffin P., Abedin S., Pishko A., Michel J.J., Rosenkranz M.E., Reed A.M., Kietz D.A, & Vallejo A.N. (2013). Premature cell senescence and T cell receptor-independent activation of CD8T cells in Juvenile Idiopathic Arthritis. Arthritis and rheumatism, 65(8), 2201-2210.

Publication 2013

5-(6)-carboxyfluorescein diacetate succinimidyl ester alpha-beta T-Cell Receptor Biological Assay Cell Culture Techniques Cells Cytokine Flow Cytometry Golgi Apparatus Granzymes IL2RA protein, human Immunoglobulin Isotypes Interleukin-2 Muromonab-CD3 Phenotype Proleukin Stem Cells T-Lymphocyte Tissues Tyrosine

Cytokine Profiling and Signaling in Immune Cells

Cited 4 times

Human and mouse IL-22 ELISA kits were obtained from Antigenix America. CCL20 ELISA kit, recombinant CCL20, IL-23 ELISA kit, anti-human IL-22 antibody for intracellular staining and anti-mouse NKp46 were obtained by R&D. IL-22 and IL-1β were purchased from Peprotec. IL-22 was used at 80 ng/ml for stimulation of epithelial cells. IL-23 was used at 40ng/ml for stimulation of NK cells, unless otherwise stated. IL-6, IL-15, IL-26, mouse and human IL-23 were purchased from R&D. Anti-IL-17A antibody was obtained from eBioscience. IL-12p70 and anti-IFN-γ, -TNF-α, -GM-CSF, -LIF, -perforin, -granzyme, -CCR6, -CD103, -pSTAT3, -pSTAT1, -NK1.1, -mouse CD3 and -mouse CD19 antibodies were purchased from Pharmingen. Anti-BCL-3, -BCL-6, -MCL-1 and -actin antibodies for immunoblotting were obtained from Santa Cruz Biotechnology. IL-10 was quantified in cell culture supernatants with the CBA human inflammation kit (Pharmingen). Anti-NKp44 and -CD96 antibodies were produced and conjugated in our laboratory.

Cella M., Fuchs A., Vermi W., Facchetti F., Otero K., Lennerz J.K., Doherty J.M., Mills J.C, & Colonna M. (2008). A human NK cell subset provides an innate source of IL-22 for mucosal immunity. Nature, 457(7230), 722-725.

Publication 2008

Actins alpha HML-1 Antibodies Antibodies, Anti-Idiotypic BCL3 protein, human BCL6 protein, human CCL20 protein, human CCR6 protein, human Cell Culture Techniques Enzyme-Linked Immunosorbent Assay Epithelial Cells Granulocyte-Macrophage Colony-Stimulating Factor Granzymes Homo sapiens IL10 protein, human IL17A protein, human IL22 protein, human Inflammation Interferon Type II Interleukin-1 beta Interleukin-12 Interleukin-15 Mus Natural Killer Cells NCR1 protein, human NCR2 protein, human Perforin Protoplasm Tumor Necrosis Factor-alpha

Immunophenotyping of human NK cells

Cited 3 times

Immunophenotyping of human NK cells was performed using flow cytometry analyses. Cryopreserved peripheral blood mononuclear cells (PBMC) collected from 15 healthy normal adult donors were thawed and incubated overnight (18 h) in RPMI 1640 medium supplemented with 10% FBS, or in RPMI 1640 medium supplemented with 10% FBS and 10 ng/mL recombinant human IL-15 (Miltenyi Biotec, GmbH). The cells were then washed with PBS and stained with a viability marker (Fixable Aqua Dead Cell Stain Kit, Thermo Fisher Scientific, San Diego, CA) prior to surface and intracellular staining with fluorescently conjugated Abs using standard techniques. The staining panel used to identify NK cell subsets and phenotypes was based on the Optimized Multicolor Immunofluorescence Panel (OMIP) described previously [OMIP-007, (33 (link))]. Fluorescently conjugated Abs used for surface staining were: PE-TR-CD3, clone S4.1, Thermo Fisher Scientific; APC-H7-CD4, clone SK3, BD Biosciences, San Jose, CA; PE-Cy5-CD14, clone Tuk4, Thermo Fisher Scientific; PE-Cy5-CD19, clone SJ25-C1, Thermo Fisher Scientific; PacificBlue-CD16, clone 3G8, BD Biosciences; PE-Cy7-CD56, clone NCAM16.2, BD Biosciences; BV606-CD62L, clone DREG-56, Biolegend, San Diego, CA; FITC-HLA-DR, clone G46-6, BD Biosciences; APC-CD57, clone HCD57, Biolegend; and BV785-CD69, clone FN50, Biolegend. Intracellular staining was performed with BV711-Perforin, clone dG9, Biolegend, and PE-Granzyme B, clone GB11, BD Biosciences. Quantum™ Simply Cellular^® beads (Bangs Laboratories, Inc., Fishers, Indiana) were used to determine the Ab binding capacity (ABC) of perforin and granzyme within cells according to the manufacturer's recommended procedure. Data analysis was performed using FlowJo software (v10.5.3).

Fisher L., Zinter M., Stanfield-Oakley S., Carpp L.N., Edwards R.W., Denny T., Moodie Z., Laher F., Bekker L.G., McElrath M.J., Gilbert P.B., Corey L., Tomaras G., Pollara J, & Ferrari G. (2019). Vaccine-Induced Antibodies Mediate Higher Antibody-Dependent Cellular Cytotoxicity After Interleukin-15 Pretreatment of Natural Killer Effector Cells. Frontiers in Immunology, 10, 2741.

Full text: Click here

Publication 2019

Adult Cells Clone Cells Donors Flow Cytometry Fluorescein-5-isothiocyanate Fluorescent Antibody Technique Granzymes GZMB protein, human HLA-DR Antigens Homo sapiens Interleukin-15 Natural Killer Cells PBMC Peripheral Blood Mononuclear Cells Perforin Phenotype Protoplasm SELL protein, human Stains

Most recents protocols related to «Granzymes»

T Cell Surface Marker Detection Protocol

For the detection of cell surface marker, 2 × 10⁵ T cells were washed and probed with antibodies in FACS buffer (1% bovine serum albumin [BSA] in DPBS) for 20 min at 4°C. To exclude the dead cell population, cells were stained with the fixable vitality dye, eFluor 780 (65-0865-14; eBioscience) for 10 min at 25°C. After being washed with FACS buffer, the cells were probed with chloroform-conjugated specific antibodies. NY-ESO-1-targeting TCR expression was determined with allophycocyanin (APC)-conjugated TCR Vβ13.1 antibody (362410; BioLegend), and CD3ϵ and TCR α/β were detected with Alexa Fluor^® 488 anti-human TCR α/β antibody (306712; BioLegend) and Brilliant Violet (BV)-421-conjugated CD3 antibody (300434; BioLegend). PD-1 upregulation was analyzed using BV421-conjugated CD279 antibody (367422; BioLegend). tLNGFR expression was analyzed using APC-conjugated CD271 antibody (130-113-418; Miltenyi Biotec). To detect CD4 and CD8, PerCP-CP/Cyanine5.5-conjugated CD4 antibody (357414; BioLegend) and APC-conjugated CD8 antibody (344722; BioLegend) were used. To determine differentiation status, BV421-conjugated CD45RO antibody (562641; BD Biosciences) and PE-conjugated CD197 antibody (560765; BD Biosciences) were used. To analyze intracellular proteins, surface-stained cells were fixed and permeabilized using a Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences). After being washed with intracellular staining buffer (1% BSA, 0.1% sodium azide, and 0.1% saponin in DPBS), the cells were probed with following antibodies. IFN-γ secretion was detected with BV711-conjugated IFN- γ antibody (564039; BD Bioscience). The releases of granzyme B (GzmB) and perforin were detected using APC-conjugated GzmB antibody (396408; BioLegend) and PE-conjugated perforin antibody (353304; BioLegend), respectively. The percentage of proliferative cells after repeated stimulation was analyzed with APC-conjugated Ki67 antibody (556027; BD Biosciences). All flow cytometry data were acquired with a BD LSRFortessa X-20 Cell Analyzer (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).

Kim S., Park C.I., Lee S., Choi H.R, & Kim C.H. (2023). Reprogramming of IL-12 secretion in the PDCD1 locus improves the anti-tumor activity of NY-ESO-1 TCR-T cells. Frontiers in Immunology, 14, 1062365.

Full text: Click here

Publication 2023

alexa fluor 488 allophycocyanin allophycocyanin B Antibodies Antibodies, Anti-Idiotypic Buffers CD45RO Antigens Cells Chloroform Flow Cytometry Granzymes GZMB protein, human Homo sapiens Immunoglobulins Interferon Type II NGFR protein, human Patient Discharge PDCD1 protein, human Perforin Proteins Protoplasm Saponin secretion Serum Albumin, Bovine Sodium Azide T-Lymphocyte Transcriptional Activation Viola

Single-cell RNA-seq Immune Cell Profiling

Major cell types were annotated based on expression of canonical marker genes (AMp: CD68, CD163, FABP4, MARCO; monocytes: VCAN, FCN1; mDC: CD1C, CD1E, FLT3, LGALS2; cDC1: FLT3, LGALS2, CLEC9A, XCR1; neutrophils: FCGR3B, CXCL8, CSF3R; G0S2; Mast cells: TPSAB1, TPSB2, MS4A2, GATA2; T-cells: CD3D, CD3E, IL32; CD4 T-cells: CD4; CD8 T-cells: CD8A, CD8B; Tregs: FOXP3, IL2RA; NK-cells: TRDC, NKG7, KLRC1, XCL1, XCL2, KLRF1; B-cells: MS4A1, SPIB; Plasma-cells: JCHAIN, SDC1, MZB1, TNFRSF17, XBP1; Fibroblasts: COL3A1, COL2A1, MFAP5; proliferating cells: MKI67, TOP2A). T-cell subpopulations were annotated upon careful assessment of expressed genes. particularly, expression of canonical lineage and cell state markers was interrogated (Th1: IFNG, TNF, TBX21, CXCR3; Th17: IL17A, CCL20, RORC, CCR6, KLRB1; central memory T-cell (TCM): CCR7, TCF7, S1PR1, IL7R, SELL; naive T-cells: CCR7, TCF7, LEF1; MAIT: SLC4A10, ME1; cytotoxic lymphocytes (CTL): Granzymes, PRF1; CD4 CTL: CD4, CCL4, CCL5, PRF1; IFN-responsive: ISG15, MX1, RSAD2). Macrophage subpopulations were annotated in an analogous fashion (CD163/LGMN Mp: SPP1, LGMN; pro-inflammatory AMp: CCL3, CCL4, CCL20, TNFAIP6, SOD2; metallothionein AMp: metallothioneins, e.g. MT1G, MT2A, MT1X; IFN-responsive: ISG15, MX1, RSAD2; Foam cells: LDLR, SQLE, HMGCR, MSMO1).

Leipold A.M., Werner R.A., Düll J., Jung P., John M., Stanojkovska E., Zhou X., Hornburger H., Ruckdeschel A., Dietrich O., Imdahl F., Krammer T., Knop S., Rosenwald A., Buck A., Sander L.E., Einsele H., Kortüm K.M., Saliba A.E, & Rasche L. (2023). Th17.1 cell driven sarcoidosis-like inflammation after anti-BCMA CAR T cells in multiple myeloma. Leukemia, 37(3), 650-658.

Full text: Click here

Publication 2023

B-Lymphocytes CCL3 protein, human CCL4 protein, human CCL5 protein, human CCL20 protein, human CCR6 protein, human CCXCR1 receptor, human CD1C protein, human CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes CD163 protein, human Cells Central Memory T Cells CSF3R protein, human CXCL8 protein, human CXCR3 protein, human FABP4 protein, human Fibroblasts FLT3 protein, human Foam Cells Galectin 2 GATA2 protein, human Gene Expression Genes Granzymes HMGCR protein, human IL2RA protein, human IL32 protein, human Inflammation Interferon Type II Interleukin-17A Interleukin 7 Receptor KLRB1 protein, human LDLR protein, human LEF1 protein, human Lymphocyte Macrophage Mast Cell Metallothionein MKI67 protein, human Monocytes Natural Killer Cells Neutrophil Plasma Cells Population Group PRF1 protein, human SELL protein, human SOD2 protein, human SPP1 protein, human T-Lymphocyte TBX21 protein, human TCF7 protein, human Th17 Cells TNFRSF17 protein, human TOP2A protein, human X-box binding protein 1, human XCL1 protein, human XCL2 protein, human

Immunosuppressive Pathway Targeting Protocol

N₃-PEG₂₀₀₀-ACA was purchased from MeloPEG (Shengzhen, China). 1,4-butanediol diacrylate (HDD), 4,4’-trimethyldipiperidine (TDP), tris (3-hydroxypropyltriazolyl methyl) amine (THPTA), fructose-6-phosphate (F6P), IR-780 were purchased from Aladdin (Shanghai, China). BAY-876 and UDP-GlcNAc were purchased from MedChemExpress (Shanghai, China). Annexin V-FITC apoptosis detection kit, DAPI, and TUNEL detection kit were purchased from Beyotime (Shanghai, China). ELISA kit of IFN-γ, TNF-α, CXCL-10 and granzyme were purchased from ABclonal (Wuhan, China). Fructose-6-Phosphate (F6P) Assay Kit was purchased from Sigma-Aldrich. Red blood cell lysis buffer and Glycerol anhydrous were obtained from Solarbio (Beijing, China). Mouse-PD1 protein, Mouse-biotin-PD-L1 and Mouse-biotin-CTLA-4 were purchased from ACROBiosystems (Beijing, China). Streptavidin-coated magnetic beads were purchased from BEAVER (Suzhou, China). DNA was purchased from Sangon (Shanghai, China), and the corresponding sequence information was provided in Supplementary Table 1. siNC and siPD-L1 were purchased from Biosyntech (Suzhou, China), and the corresponding sequence information was provided in Supplementary Table 2. The information of antibodies was provided in Supplementary Tables 3 and 4.

Ren X., Cheng Z., He J., Yao X., Liu Y., Cai K., Li M., Hu Y, & Luo Z. (2023). Inhibition of glycolysis-driven immunosuppression with a nano-assembly enhances response to immune checkpoint blockade therapy in triple negative breast cancer. Nature Communications, 14, 7021.

Full text: Click here

Publication 2023

Antibodies Apoptosis BAY-876 Beavers Biological Assay Biotin bis(tri-n-hexylsiloxy)(2,3-naphthalocyaninato)silicon Buffers butanediol diacrylate CD274 protein, human CTLA4 protein, human DAPI Enzyme-Linked Immunosorbent Assay Erythrocytes FITC-annexin A5 fructose-6-phosphate Glycerin Granzymes In Situ Nick-End Labeling Interferon Type II IR 780 methylamine Mus Pdcd1 protein, mouse Streptavidin Tromethamine Tumor Necrosis Factor-alpha

Antigen-specific T-cell Clone Generation

Melan-A-specific and gp100-specific T-cell clones were generated by limiting dilution from pre-sensitized antigen (Ag)-specific T-cell lines, as described [23 (link)], and periodically re-stimulated in RPMI 1640 supplemented with 10% human serum (HS, noncommercial, prepared from healthy donors), 1 μg/ml PHA (Roche, 11,082,132,001), 25 U/ml recombinant IL-2 (rIL-2) (Miltenyi Biotec, 130–097-746), and 1 × 10⁶/ml irradiated allogenic PBMC as feeder cells. T-cell clones were used 13–15 d after stimulation. Ag-specificity of T-cell clones was assessed and periodically confirmed using anti-CD8-FITC and HLA-A*0201/PE-Melan-A (ELAGIGILTV) and HLA-A*0201/PE-gp100 (ITDQVPFSV) tetramer staining (see Table S2), performed for 30 min at room temperature (RT). Dead cells were excluded using propidium iodide staining (MP Biomedicals, 195,458). For intracellular staining of lytic molecules, Melan-A-specific and gp100-specific (n = 24) T-cell clones, isolated from 5 melanoma patients, were co-cultured with related (Melan-A⁺gp100⁺, Mel3) or unrelated (Melan-A⁻gp100⁻, Mel2) HLA/Ag melanoma cell lines, for 4–5 h at 37°C in the presence of protein transport inhibitor GolgiStop (BD, 554,724). T-cell clones were collected, and intracellular staining was performed using Intrasure kit (BD, 641,778) according to the manufacturer's instruction. Cells were then incubated for 30 min at RT with anti- PerCP-Cy5.5- tumor necrosis factor (TNF)-α, PE-Cy7-interferon (IFN)-γ and Alexa Fluor647-granzyme (GrzB) mAbs (see Table S2). Lytic activity was assessed in a standard 4-h ⁵¹Cr release assay. Mel2 and Mel3 melanoma cell lines were used as target cells and cytotoxicity was performed by incubating ⁵¹Cr-labeled target cells with effector cells (n = 26) at an effector: ratio (E:T) ratio of 20:1.

Palermo B., Franzese O., Frisullo G., D’Ambrosio L., Panetta M., Campo G., D’Andrea D., Sperduti I., De Nicola F., Goeman F., Gallina F., Visca P., Facciolo F, & Nisticò P. (2023). CD28/PD1 co-expression: dual impact on CD8+ T cells in peripheral blood and tumor tissue, and its significance in NSCLC patients' survival and ICB response. Journal of Experimental & Clinical Cancer Research : CR, 42, 287.

Full text: Click here

Publication 2023

Antigens Biological Assay Cell Lines Cells Clone Cells CY5.5 cyanine dye Cytotoxin Donors Feeder Cells Fluorescein-5-isothiocyanate Granzymes HLA Antigens Homo sapiens Interferons Interferon Type II Melanoma MLANA protein, human Monoclonal Antibodies Patients Propidium Iodide Protein Transport Protoplasm Serum T-Cell Specificity T-Lymphocyte Technique, Dilution Tetrameres Tumor Necrosis Factor-alpha

Tumor Immune Cell Analysis by Flow Cytometry

Prepared for single cell suspensions: for the murine samples, subcutaneous tumor mice or bone tumor mice were sacrificed by CO₂. Blood samples were drawn from tumor mice heart, and tissues of spleens, femurs, and tumors were extracted and minced by scissor. Femurs and tumors need to be digested with collagenase IV (Sigma-Aldrich, 0.2%) for 30 min at 37 °C and were terminated by RMPI 1640 (serum). Suspensions was strained through a 70-μm cell strainer to obtain a single-cell fluid. Red blood cells were lysed by lysis buffer (BD, 555899).
Antibody incubation and examination: 1 × 10 ⁶ cells from single cell suspensions were labeled with fluorescence-conjugated antibodies: APC-CD11b (Clone: M1/70; eBioscience™), PE-Ly6G (Clone: 1A8; MilliporeSigma™), Fitc-CD4 (Clone: GK1.5; eBioscience™), APC-CD8 (Clone: 53–6.7; R&D Systems™), PE-Perforin (Clone: eBioOMAK-D; eBioscience™), PerCP-5.5-Granzyme (Clone: NGZB; eBioscience™), PerCP-5.5-Arginase (Polyclonal, Novus Biologicals™), and PE-iNOS (Clone: CXNFT, eBioscience™), PE-EpCAM (Clone: EBA-1, BD Biosciences). Cells were staining for 30 min at 4 °C and washed with PBS twice before examined. For intracellular protein markers (arginase, iNOS, granzyme, and perforin), cells were conducted with fixation/permeabilization procedure following the instruction (BD Biosciences, 555028) before being stained with fluorescently labeled antibodies for 30 min. Cells labeled with fluorescence-conjugated antibodies were then examined with BD LSR Fortessa Analyzer and analyzed with FlowJo v10.6.2. Prostate cancer cells labeled with PE-EpCAM were isolated with FACS Aria sorting system for following experiments.

Chen Z., Yang X., Chen Z., Li M., Wang W., Yang R., Wang Z., Ma Y., Xu Y., Ao S., Liang L., Cai C., Wang C., Deng T., Gu D., Zhou H, & Zeng G. (2023). A new histone deacetylase inhibitor remodels the tumor microenvironment by deletion of polymorphonuclear myeloid-derived suppressor cells and sensitizes prostate cancer to immunotherapy. BMC Medicine, 21, 402.

Full text: Click here

Publication 2023

Antibodies Arginase Biological Factors BLOOD Buffers Cells Clone Cells Collagenase Erythrocytes Femur Fluorescein-5-isothiocyanate Fluorescent Antibody Technique Granzymes Heart Neoplasm Immunoglobulins Intracavitary Tumors of the Heart ITGAM protein, human Mus Neoplasms Neoplasms, Bone NOS2A protein, human Novus NRG1 protein, human Perforin Prostate Cancer Proteins Protoplasm Serum TACSTD1 protein, human Tissues

Top products related to «Granzymes»

Cytofix cytoperm kit by BD

Sourced in United States, Germany, France, United Kingdom, China, Italy, Belgium, Canada, Australia

The Cytofix/Cytoperm kit is a laboratory product designed for fixing and permeabilizing cells. It provides the necessary solutions for the preparation of samples prior to intracellular staining and flow cytometric analysis.

Brefeldin a by Merck Group

Sourced in United States, Germany, United Kingdom, Macao, Canada, Switzerland, France, Japan, Sao Tome and Principe, Israel, Italy, Chile

Brefeldin A is a fungal metabolite that inhibits the function of Golgi apparatus in eukaryotic cells. It acts by blocking the exchange of materials between the endoplasmic reticulum and Golgi compartments, leading to the collapse of the Golgi structure.

Facsdiva software by BD

Sourced in United States, Germany, United Kingdom, France, Canada, Belgium, Australia, Italy, Spain, Switzerland, China, Netherlands, Finland, Japan, Jersey, Lao People's Democratic Republic

FACSDiva software is a user-friendly flow cytometry analysis and data management platform. It provides intuitive tools for data acquisition, analysis, and reporting. The software enables researchers to efficiently process and interpret flow cytometry data.

Lsrfortessa by BD

Sourced in United States, United Kingdom, Germany, France, Canada, Australia, Belgium, China, Uruguay, Japan, Sweden, Switzerland, Cameroon

The LSRFortessa is a flow cytometer designed for multiparameter analysis of cells and other particles. It features a compact design and offers a range of configurations to meet various research needs. The LSRFortessa provides high-resolution data acquisition and analysis capabilities.

Anti ifn γ pe cy7 by Thermo Fisher Scientific

Sourced in United States

The Anti-IFN-γ-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the interferon-gamma (IFN-γ) protein. It is designed for use in flow cytometry applications to detect and quantify IFN-γ-producing cells.

Facscalibur flow cytometer by BD

Sourced in United States, Germany, United Kingdom, China, Canada, Japan, Belgium, France, Spain, Italy, Australia, Finland, Poland, Switzerland, Cameroon, Uruguay, Denmark, Jersey, Moldova, Republic of, Singapore, India, Brazil

The FACSCalibur flow cytometer is a compact and versatile instrument designed for multiparameter analysis of cells and particles. It employs laser-based technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or other particles as they flow in a fluid stream. The FACSCalibur can detect and quantify a wide range of cellular properties, making it a valuable tool for various applications in biology, immunology, and clinical research.

Anti granzyme a percp cy5 by BioLegend

Anti-granzyme A-PerCp/Cy5.5 is a fluorescently labeled antibody that binds to the granzyme A protein. Granzyme A is a serine protease found in the cytotoxic granules of natural killer cells and cytotoxic T cells, and is involved in inducing cell death. The PerCp/Cy5.5 fluorescent label allows for detection and quantification of cells expressing granzyme A.

Ionomycin by Merck Group

Sourced in United States, Germany, United Kingdom, Macao, France, Italy, China, Canada, Switzerland, Sao Tome and Principe, Australia, Japan, Belgium, Denmark, Netherlands, Israel, Chile, Spain

Ionomycin is a laboratory reagent used in cell biology research. It functions as a calcium ionophore, facilitating the transport of calcium ions across cell membranes. Ionomycin is commonly used to study calcium-dependent signaling pathways and cellular processes.

Cell stimulation cocktail by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Canada

The Cell Stimulation Cocktail is a laboratory reagent used to activate and stimulate cells in culture. It contains a combination of chemical compounds that induce cellular responses, such as cytokine production or signal transduction pathways. The specific composition and concentration of the cocktail can vary depending on the intended application and experimental requirements.

Anti cd3 phycoerythrin cyanine 7 pe cy7 by Beckman Coulter

Sourced in United States

Anti-CD3 phycoerythrin-cyanine 7 (PE-Cy7) is a fluorescent-conjugated antibody that binds to the CD3 cell surface antigen. It is a reagent used for the identification and enumeration of T cells in flow cytometry applications.

More about "Granzymes"

Granzymes are a family of serine proteases found in the cytotoxic granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs).
These powerful enzymes play a crucial role in the induction of apoptosis, or programmed cell death, in target cells during the cell-mediated immune response.
Understanding the mechanisms of granzyme function is essential for developing effective therapies targeting these important immune effector molecules.
Granzymes have been implicated in a variety of physiological and pathological processes, including tumor suppression, autoimmune disorders, and inflammation.
Researchers can utilize tools like the Cytofix/Cytoperm kit, Brefeldin A, and FACSDiva software to study the expression and activity of granzymes in immune cells.
Flow cytometry techniques using instruments like the LSRFortessa and FACSCalibur can help quantify and analyze granzyme levels.
Specific antibodies, such as Anti-IFN-γ-PE-Cy7 and Anti-granzyme A-PerCp/Cy5.5, can be employed to identify and characterize granzyme-producing cells.
Additionally, strategies like stimulating immune cells with Ionomycin and Cell Stimulation Cocktail, or activating them via Anti-CD3 phycoerythrin-cyanine 7 (PE-Cy7), can provide insights into the regulation and function of granzymes in various immune responses.
By leveraging these tools and techniques, researchers can deepen their understanding of granzyme biology and explore new avenues for therapeutic interventions targeting these crucial immune effector molecules.