Two-color flow cytometric analysis of Granzyme A expression in C57BL/6 mouse splenocytes. Mouse spleen cells were treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes, washed, and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Mouse Anti-Mouse NK1.1 (Cat. No. 550627/561117) before being fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by permeabilization with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained with either PE Mouse IgG2a, κ Isotype Control (Cat. No. 555058; Left Plot) or PE Mouse Anti-Mouse Granzyme A (Cat. No. 567849; Right Plot) at 0.25 μg/test. The pseudocolor density plot showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus NK1.1 were derived from gated events with the forward and side light-scatter characteristics of intact splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Two-color flow cytometric analysis of Granzyme A expression in BALB/c mouse splenocytes. Mouse spleen cells were cultured (3 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057) and soluble NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294/ 567110) antibodies. The activated cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and stained with APC Mouse Anti-Mouse CD8a antibody (Cat. No. 553035/561093) before being fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by permeabilization with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained with either PE Mouse IgG2a, κ Isotype Control (Cat. No. 555058; Left Plot) or PE Mouse Anti-Mouse Granzyme A (Cat. No. 567849; Right Plot) at 0.25 μg/test. The pseudocolor density plots showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus CD8a were derived from gated events with the forward and side light-scatter characteristics of intact activated splenocytes. Flow cytometry and data analysis were performed using a BD FACSCelesta™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
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Two-color flow cytometric analysis of Granzyme A expression in C57BL/6 mouse splenocytes. Mouse spleen cells were treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes, washed, and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Mouse Anti-Mouse NK1.1 (Cat. No. 550627/561117) before being fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by permeabilization with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained with either PE Mouse IgG2a, κ Isotype Control (Cat. No. 555058; Left Plot) or PE Mouse Anti-Mouse Granzyme A (Cat. No. 567849; Right Plot) at 0.25 μg/test. The pseudocolor density plot showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus NK1.1 were derived from gated events with the forward and side light-scatter characteristics of intact splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Two-color flow cytometric analysis of Granzyme A expression in BALB/c mouse splenocytes. Mouse spleen cells were cultured (3 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057) and soluble NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294/ 567110) antibodies. The activated cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and stained with APC Mouse Anti-Mouse CD8a antibody (Cat. No. 553035/561093) before being fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by permeabilization with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained with either PE Mouse IgG2a, κ Isotype Control (Cat. No. 555058; Left Plot) or PE Mouse Anti-Mouse Granzyme A (Cat. No. 567849; Right Plot) at 0.25 μg/test. The pseudocolor density plots showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus CD8a were derived from gated events with the forward and side light-scatter characteristics of intact activated splenocytes. Flow cytometry and data analysis were performed using a BD FACSCelesta™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
製品詳細
BD Pharmingen™
Gzma; Ctla3;Ctla-3;TSP1;Hf;TSP-1;SE1
Mouse (QC Testing)
Mouse IgG2b, κ
Full-length Recombinant Mouse Granzyme A
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unconjugated antibody and free dye were removed.
推奨アッセイ手順
BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
関連製品
Stain Buffer (FBS) RUO
サイズ
500 mL
カタログ番号
554656
Stain Buffer (BSA) RUO
サイズ
500 mL
カタログ番号
554657
Lysing Buffer RUO
サイズ
100 mL
カタログ番号
555899
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) RUO
サイズ
0.1 mg
カタログ番号
553141
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) RUO
サイズ
0.5 mg
カタログ番号
553142
Fixation Buffer RUO
サイズ
100 mL
カタログ番号
554655
567849 Rev. 1
抗体の詳細
3G8.5
The 3G8.5 monoclonal antibody specifically recognizes Granzyme A which is also known as T cell-specific serine protease 1 (TSP-1), Cytotoxic T-lymphocyte-associated serine esterase-3 (CTLA-3), or Fragmentin-1. Granzyme A is a serine protease that cleaves various peptide substrates after lysine or arginine residues and is encoded by Gzma (granzyme A). This effector protease presents as a homodimer that is stored in the cytosolic granules of cytotoxic T cells and NK cells. Granzyme A plays an important role in inflammatory and cell-mediated cytotoxic responses against malignant target cells and cells infected with viruses or bacteria. It is released from effector cells by granule exocytosis and enters target cells at immunological synapses. Therein, Granzyme A activates caspase-independent target cell death that is mainly characterized by the generation of single-stranded DNA nicks, loss of mitochondrial transmembrane potential, and loss of cell membrane integrity.
567849 Rev. 1
フォーマットの詳細
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
567849 Rev.1
引用＆参考文献
Development References (7)
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Bots M, Medema JP. Granzymes at a glance.. J Cell Sci. 2006; 119(Pt 24):5011-4. (Biology). View Reference
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Cao X, Cai SF, Fehniger TA, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance.. Immunity. 2007; 27(4):635-46. (Immunogen: Flow cytometry). View Reference
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Fehniger TA, Cai SF, Cao X, et al. Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs.. Immunity. 2007; 26(6):798-811. (Clone-specific: Flow cytometry). View Reference
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Gotthardt D, Prchal-Murphy M, Seillet C, et al. NK cell development in bone marrow and liver: site matters.. Genes Immun. 2014; 15(8):584-7. (Clone-specific: Flow cytometry). View Reference
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Grossman WJ, Verbsky JW, Tollefsen BL, Kemper C, Atkinson JP, Ley TJ. Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells.. Blood. 2004; 104(9):2840-8. (Biology). View Reference
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Martinvalet D, Dykxhoorn DM, Ferrini R, Lieberman J. Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death.. Cell. 2008; 133(4):681-92. (Biology). View Reference
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Moffat JM, Gebhardt T, Doherty PC, Turner SJ, Mintern JD. Granzyme A expression reveals distinct cytolytic CTL subsets following influenza A virus infection.. Eur J Immunol. 2009; 39(5):1203-10. (Clone-specific: Flow cytometry). View Reference
567849 Rev. 1
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
