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Two-color flow cytometric analysis of Granzyme B expression in Human peripheral blood lymphocytes. Human whole blood was fixed with BD Lyse/Fix Buffer (Cat. No. 558049). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with BD Horizon™ BUV395 Mouse Anti-Human CD8 antibody (Cat. No. 563795) and with either BD Horizon™ RB670 Mouse IgG1, κ Isotype Control (Cat. No. 571784; Left Plot) or BD Horizon™ RB670 Mouse Anti-Human Granzyme B antibody (Cat. No. 571919/571920; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of Granzyme B (or Ig Isotype control staining) versus CD8 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer and FlowJo™ Software..
BD Horizon™ RB670 Mouse Anti-Human Granzyme B
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Preparation And Storage
Recommended Assay Procedures
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.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
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Companion Products
The GB11 antibody specifically reacts with human granzyme B, a serine protease of approximately 32 kDa. Granzyme B is stored in the granules of cytotoxic T lymphocytes and NK cells along with the pore-forming protein perforin. In the classic model of target cell lysis, perforins create holes in the target cell membrane allowing entrance of granzymes. Granzyme B has been shown to act on specific substrates including caspase-3, -7, -9, and -10 which in turn give rise to enzymes that mediate apoptosis. Granzyme B may also be involved in the hydrolysis of extracellular matrix components. Detectable levels of granzyme B have been detected in sera from healthy volunteers. The immunogen used to generate the GB11 hybridoma was human granzyme B isolated from an NK cell line.
Development References (8)
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Hamann D, Baars PA, Rep MH. Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med. 1997; 186(9):1407-1418. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Poe M, Blake JT, Boulton DA. Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity. J Biol Chem. 1991; 266(1):98-103. (Biology). View Reference
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Ronday HK, van der Laan WH, Tak PP et al. Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis. Rheumatology (Oxford). 2001; 40:55-61. (Biology). View Reference
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Smyth MJ, Kelly JM, Sutton VR et al. Unlocking the secrets of cytotoxic granule proteins. J Leukoc Biol. 2001; 70:18-29. (Biology). View Reference
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Spaeny-Dekking EH, Hanna WL, Wolbink AM et al. Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo. J Immunol. 1998; 160:3610. (Immunogen: ELISA, Radioimmunoassay). View Reference
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Trapani JA, Klein JL, White PC, and Dupont B. Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes. Proc Natl Acad Sci U S A. 1988; 5:6924-6928. (Biology). View Reference
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Trapani JA, Smyth MJ, Apostolidis VA, Dawson M, and Browne KA. Granule serine proteases are normal nuclear constituents of natural killer cells. J Biol Chem. 1994; 269:18359-18365. (Biology). View Reference
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Wever PC, Van Der Vliet HJ, Spaeny LH . The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells. Immunology. 1998; 93(3):383-389. (Immunogen: ELISA, Flow cytometry, Immunoprecipitation, Intracellular Staining/Flow Cytometry, Radioimmunoassay). View Reference
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