Granzyme A is a member of the granzyme family of the serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Granzyme A is the most abundant protease in CTL and NK cells. It induces caspase-independent cell death when introduced into target cells by perforin.1 Human Granzyme A is synthesized as a precursor (262 residues) with a signal peptide (residues 1-26), a propeptide (residues 27-28) and a mature chain (residues 29-262).2 The pro-enzyme is expressed and purified. After being activated by lysyl endopeptidase, rhGranzyme A cleaves a thioester substrate described above.
Principle of the Test:
Fixed cells are permeabilized, allowing conjugated antibodies access to proteins within the cell. Cells are initially subjected to a fixation step in order to minimize leakage of proteins out of the cell. The conjugated antibody is allowed to penetrate and bind to its target within the cell. Following a final wash, cells are analyzed on a flow cytometer. Flow cytometric analysis of fluorescein conjugates will generate a signal, which can be detected using 488 nm wavelength laser excitation and monitoring emitted fluorescence with a detector, optimized to collect peak emissions at 515-545nm.
Intracellular staining antibodies are designed for multiparameter flow cytometric analysis of cells. To stain for surface proteins (e.g. CD3, CD4, CD8) in addition to the intracellular protein, we recommend that the investigator determine whether the fixation and permeabilization steps adversely affect the surface protein. If so, surface staining of cells prior to fixation and permeabilization is recommended.
For intracellular staining, cells must first be fixed and permeabilized. Use of 4% paraformaldehyde in PBS as a fixative is recommended. Other formulations or tissue fixatives may affect the staining properties of the monoclonal antibody. For permeabilization, 0.1% saponin in a balanced salt solution is effective in facilitating antibody entry into cells. Due to the reversible nature of cell membrane permeabilization, saponin must be included in all buffers used (i.e. both the staining and washing steps).
Additional Reagents Required:
Paraformaldehyde Fixative-Dissolve 4.0 g of paraformaldehyde in 100 ml of sterile PBS (10 mM phosphate buffered saline, pH 7.4) by heating the solution at 56 degrees C for about 1 hour. All solids must be fully dissolved prior to use. Store buffer at 2 degrees -8 degrees C, protected from light, for no longer than 2 weeks.
SAP buffer-Prepare a sterile solution containing 0.1% (w/v) saponin, 0.05% (w/v) NaN
1. Harvest cells, and wash twice in cold HBSS or PBS by spinning at 200 x g for 7 minutes.
3 in Hanks