Two-color flow cytometric analysis of Granzyme B expression in Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656] and permeabilized with and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD8 antibody (Cat. No. 561421/555369/561953/561952) and with either BD Horizon™ RB780 Mouse IgG1 Isotype Control (Cat. No. 568532; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human Granzyme B antibody (Cat. No. 568705/568706; 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 System and FlowJo™ Software.
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Two-color flow cytometric analysis of Granzyme B expression in Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656] and permeabilized with and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD8 antibody (Cat. No. 561421/555369/561953/561952) and with either BD Horizon™ RB780 Mouse IgG1 Isotype Control (Cat. No. 568532; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human Granzyme B antibody (Cat. No. 568705/568706; 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 System and FlowJo™ Software.
Product Details
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
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
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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).
- An isotype control should be used at the same concentration as the antibody of interest.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
APC Mouse Anti-Human CD8 RUO
Size
500 Tests
Cat No.
561953
RB780 Mouse IgG1, κ Isotype Control RUO
Size
50 µg
Cat No.
568532
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568706 Rev. 2
Antibody Details
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.
568706 Rev. 2
Format Details
RB780
The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.
RB780
Blue 488 nm
498 nm
781 nm
568706 Rev.2
Citations & References
Development References (8)
-
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
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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
-
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
-
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
568706 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
