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R718 Mouse Anti-Human Granzyme A
R718 Mouse Anti-Human Granzyme A

Flow cytometric analysis of Granzyme A 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 and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ R718 Mouse IgG1 Isotype Control (Cat. No. 566928; Left Plot) or BD Horizon™ R718 Mouse Anti-Human Granzyme A antibody (Cat. No. 566976; Right Plot). A bivariate pseudocolor density plot showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus side light-scatter (SSC) signals 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 LSRFortessa™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Flow cytometric analysis of Granzyme A 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 and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ R718 Mouse IgG1 Isotype Control (Cat. No. 566928; Left Plot) or BD Horizon™ R718 Mouse Anti-Human Granzyme A antibody (Cat. No. 566976; Right Plot). A bivariate pseudocolor density plot showing the correlated expression of Granzyme A (or Ig Isotype control staining) versus side light-scatter (SSC) signals 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 LSRFortessa™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Horizon™
CTL tryptase; CTLA3; GZMA; GrA; HFSP; Hanukkah factor
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human Cytotoxic T Cell Granule Proteins
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2869982
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 CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.

Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. Alexa Fluor® is a registered trademark of Life Technologies Corporation.
  5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  7. This product is provided under an Agreement between BIOTIUM and BD Biosciences. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
566976 Rev. 3
Antibody Details
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CB9

The CB9 monoclonal antibody specifically recognizes Granzyme A (GrA, GzmA or Gzm A) which is also known as CTL tryptase,

Cytotoxic T-lymphocyte-associated serine esterase-3 (CTLA3), Cytotoxic T-lymphocyte proteinase 1, Fragmentin-1, or Hanukah factor serine protease (HFSP). Granzyme A belongs to a family of effector proteases that include five different granzymes that play key roles in innate and adaptive immune responses to pathogens and transformed cells: Granzymes A, B, H, K and M. Granzyme A is a ~28 kDa neutral serine protease that is encoded by GZMA. It is expressed in the cytotoxic granules of natural killer (NK) cells, NKT cells, cytotoxic αβ T cells and γδ T cells. Granzyme A participates in innate and adaptive immune responses. After gaining access into target cells, Granzyme A cleaves various peptide substrates after lysine or arginine residues and can induce caspase‑independent cell death reflected in single-stranded DNA damage as well as rapid loss of cell membrane integrity and mitochondrial transmembrane potential.

The antibody was conjugated to BD Horizon Red 718, which has been developed exclusively for BD Biosciences as a better alternative to Alexa Fluor® 700. BD Horizon Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor® 700.

566976 Rev. 3
Format Details
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R718
The BD Horizon™ Red 718 (R718) Dye is part of the BD red family of dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 695-nm and an emission maximum (Em Max) at 718-nm. Driven by BD innovation, R718 is designed to be excited by the red laser (627–640-nm) and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). R718 is a brighter alternative to Alexa Fluor™ 700. R718 is also a bright small molecule alternative to APC-R700 with lower spread into the APC detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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R718
Red 627-640 nm
695 nm
718 nm
566976 Rev.3
Citations & References
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Development References (8)

  1. Andrade F, Roy S, Nicholson D, Thornberry N, Rosen A, Casciola-Rosen L. Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity. 1998; 8(4):451-460. (Biology). View Reference
  2. Barry M, Heibein JA, Pinkoski MJ, et al. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol Cell Biol. 2000; 20(11):3781-3794. (Biology). View Reference
  3. Beresford PJ, Kam CM, Powers JC, Lieberman J. Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them. Proc Natl Acad Sci U S A. 1997; 94(17):9285-9290. (Immunogen: Immunoprecipitation). View Reference
  4. Beresford PJ, Xia Z, Greenberg AH, Lieberman J. Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity. 1999; 10(5):585-594. (Clone-specific: Immunoprecipitation). View Reference
  5. Pinkoski MJ, Waterhouse NJ, Heibein JA, et al. Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway. J Biol Chem. 2001; 276(15):12060-12067. (Biology). View Reference
  6. Sharif-Askari E, Alam A, Rheaume E, et al. Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J. 2001; 20(12):3101-3113. (Biology). View Reference
  7. Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ. Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity. 1999; 10(5):595-605. (Biology). View Reference
  8. Trimble LA, Lieberman J. Circulating CD8 T lymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex. Blood. 1998; 91(2):585-594. (Clone-specific: Flow cytometry). View Reference
View All (8) View Less
566976 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.