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RB545 Mouse Anti-Human Granzyme B
RB545 Mouse Anti-Human Granzyme B
Two-color flow cytometric analysis of Granzyme B expression in Human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells (PBMC) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD8 antibody (Cat. No. 561953/555369/561421/561952) and with either BD Horizon™ RB545 Mouse IgG1, κ Isotype Control (Cat. No. 569284; Left Plot) or BD Horizon™ RB545 Mouse Anti-Human Granzyme B antibody (Cat. No. 569290/569291; 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.  Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ v10.8 software.
Two-color flow cytometric analysis of Granzyme B expression in Human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells (PBMC) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD8 antibody (Cat. No. 561953/555369/561421/561952) and with either BD Horizon™ RB545 Mouse IgG1, κ Isotype Control (Cat. No. 569284; Left Plot) or BD Horizon™ RB545 Mouse Anti-Human Granzyme B antibody (Cat. No. 569290/569291; 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.  Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ v10.8 software.
Product Details
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BD Horizon™
GZMB; Granzyme-2; CCPI; CGL1; CSPB; CTLA1; CTSGL1; GRB; HLP; SECT
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human Granzyme B
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl/test
3002
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 unreacted dye was removed.

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

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  3. 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.
  4. 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).
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. An isotype control should be used at the same concentration as the antibody of interest.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
  9. For U.S. patents that may apply, see bd.com/patents.
569290 Rev. 1
Antibody Details
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GB11

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.

569290 Rev. 1
Format Details
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RB545
The BD Horizon RealBlue™ 545 (RB545) fluorochrome is part of the BD family of blue (488-nm) laser dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 496-nm and an emission maximum (Em Max) at 545-nm. Driven by BD innovation, RB545 can be used on spectral cytometers and is designed to be excited by the blue (488-nm) laser with minimal excitation by the 561-nm yellow-green laser. RB545 has minimal spillover into yellow-green detectors and a brightness level similar to FITC.  Given its unique emission max, on a spectral instrument RB545 can be used simultaneously with FITC and PE to provide an additional color off of the blue laser. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
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RB545
Blue 488 nm
496 nm
545 nm
569290 Rev.1
Citations & References
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Development References (8)

  1. 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: Flow cytometry). View Reference
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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, Immunocytochemistry (cytospins), Immunoprecipitation, Radioimmunoassay). View Reference
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569290 Rev. 1

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