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Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 488 Conjugate)

Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 488 Conjugate) #33359


H M Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left column) or treated with anti-CD3 plus anti-CD28 (10 μg/ml, 72 hr; right column), using Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 488 Conjugate) (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (bottom row), and co-stained with a CD8 Antibody.

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total Granzyme B protein.

Species Reactivity:

Human, Mouse

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to human Granzyme B protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Granzyme B (D2H2F) Rabbit mAb #17215.

Granzymes are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer (NK) cells and are key components of immune responses to pathogens and transformed cells (1). Granzymes are synthesized as zymogens and are processed into mature enzymes by cleavage of a leader sequence. They are released by exocytosis in lysosome-like granules containing perforin, a membrane pore-forming protein. Granzyme B has the strongest apoptotic activity of all the granzymes as a result of its caspase-like ability to cleave substrates at aspartic acid residues thereby activating procaspases directly and cleaving downstream caspase substrates (2,3).

  1. Trapani, J.A. (2001) Genome Biol. 2, REVIEWS 3014.
  2. Lord, S.J. et al. (2003) Immunol. Rev. 193, 31-8.
  3. Trapani, J.A. and Sutton, V.R. (2003) Curr. Opin. Immunol. 15, 533-43.
Entrez-Gene Id
Swiss-Prot Acc.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

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To Purchase # 33359S

Product Number Size Price
33359S 100 µl (50 tests) $299.00.0
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