Mechanism of Prizm™ Fluorogenic Cell Assay Kits


Mechanism_Cell_Assays.gif PrizmTM Fluorogenic Cell Assay Kits are based on cell-permeable fluorogenic substrates designed to monitor enzymatic activity in biological samples. These substrates diffuse through plasma or intracellular membranes by passive diffusion. Once the substrates are recognized by their corresponding protease, they are cleaved and the fragments produce fluorescence that can help measure the activity of the enzyme.

Choice of Fluorophore
Both excitation and emission states were selected in the visible wavelength region. The rationale for this specific dye selection is that biological materials contain molecules that both absorb and fluoresce in the UV wavelength domain; this auto-fluorescence can hinder determination of small amounts of activity present in samples and reduce the reliability of the activity determination.

Substrate Design and Computer Modeled Structure
Peptide substrates are synthesized with covalently coupled fluorophores. The latter are strategically situated away from the target sequence to ensure non-interference with proteolytic cleavage. A representative computer modeled structure for substrate is shown in the figure above where the yellow ribbon represents the peptide backbone conformation and the cyan color the attached fluorophores.

Cleavage-induced Fluorescence and Absorption changes
In the intact peptide the cyan colored fluorophores form a ground-state dimer. Peptide cleavage abolishes this dye-dye interaction and results in an increase in fluorescence and significant absorption changes.

Activation of Caspase 8 (red = cleavage of substrate)

Validation Data

Apoptosis Assays

Jurkat cells were treated with 1 uM Staurosporine (green/red) or vehicle control (black) for 4 hour at 37 degrees C and then exposed to caspase substrates for 40 minutes at 37 degrees. Flow cytometric analysis followed a single wash.

Cytotoxicity Assays

To target (Jurkat) cells labeled with TFL4, effector (NK92) cells were added at a 5:1::E:T. Cells were pelleted in the presence of the substrate and then coincubated for 1 hour at 37 degrees C. After a single wash, the fluorescence of resuspended cells was measured by flow cytometry. The upper two quadrants contain TFL4-labeled target cells and the bottom two effectors. The percentage of protease-positive target cells, as represented by the number of events in the upper right over the sum of upper right and upper left, indicates the number of Jurkat cells that have received a lethal hit from NK cells.

Publications on Cell Assay Kits:

  • B.Z. Packard, W.G. Telford, A. Komoriya, and P.A. Henkart. Granzyme B activity in target cells detects attack by cytotoxic lymphocytes. J. Immunol. 179:3812- 3820 (2007).
  • A. Kinter, J. McNally, L. Riggin, R. Jackson, G. Roby, and A.S. Fauci. Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIVinfected individuals. PNAS 104:3390-3395 (2007).
  • T. Tarasenko, H.K. Kole, A.W. Chi, M.M. Mentink-Kane, T.A. Wynn, and S. Bolland. T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses. PNAS 104(27):11382-11387 (2007).
  • M. Carlsten, N.K. Björkström, H. Norell, et al. DNAX Accessory Molecule-1 mediated recognition of freshly isolated ovarian carcinoma by resting Natural Killer cells. Cancer Research 67: 1317-1325 (2007).
  • A.L. Kinter, R. Horak, M. Sion, et al. CD25+ regulatory T Cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIVspecific CD8+ T Cells in vitro. AIDS Research and Human Retroviruses 23:438-450 (2007).
  • R. Chiarle, C. Martinengo, C. Mastini, C. Ambrogio, V. D'Escamard, G. Forni, and G..Inghirami. The anaplastic lymphoma kinase is an effective oncoantigen for lymphoma vaccination. Nat. Med. 14:676-80 (2008).
  • A. Singh, H. Nie, B. Ghosn, H. Qin, L.H. Kwak, and K. Roy. Efficient modulation of T-cell response by dual-mode, single-carrier delivery of cytokine-targeted siRNA and DNA vaccine to antigen-presenting cells. Mol. Ther. 16:2011-2021 (2008).
  • S.A. Migueles, C.M Osborne, C. Royce et al. Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control. Immunity 29:1009-21.
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