Death Be Not Proud: PhiPhiLux from OncoImmunin Inc. Offers New Insights into Apoptosis

Time-lapsed confocla microscopic images of T-cells undergoing apoptosis. The green fluroescence (a cyanine dye, which indicates a mitochondrial membrane potential) marks healthy cells, while the red fluorescence is derived from PhipPhiLux cleavage in apoptotic cells. Time-of-death determinations are not just a challenge for coroners. Researchers in the field of apoptosis are constantly striving for ways to detect the onset of cell death earlier and with greater specificity to bring them closer

September 29, 1997


Time-lapsed confocla microscopic images of T-cells undergoing apoptosis. The green fluroescence (a cyanine dye, which indicates a mitochondrial membrane potential) marks healthy cells, while the red fluorescence is derived from PhipPhiLux cleavage in apoptotic cells.
Time-of-death determinations are not just a challenge for coroners. Researchers in the field of apoptosis are constantly striving for ways to detect the onset of cell death earlier and with greater specificity to bring them closer to the true cause of cell death. Now a small company in College Park, Maryland, OncoImmunin Inc., has provided just the tool apopoticians have been seeking. Specializing in peptide-based diagnostics for cancer and infectious diseases, this company has designed a new class of fluorogenic protease substrates that gives the earliest determinations of the onset of cell death to date. PhiPhiLux, short for fluorescent-fluorescent light, incorporates the prototypical caspase-3-recognition sequence DEVD into a bifluorophore-derivitized peptide, which mimics the structural loop conformation present in native protease cleavage sites of globular proteins. The advantages of this class of protease substrates are numerous. Among them

  • they work both in solution and in cells, in common buffered solutions found in research labs and in bodily fluids encountered in clinical settings. They can be used with quartz as well as plastic cuvettes and in high throughput formats (microtiter plate, for example)

  • they have the ability to penetrate membranes, enabling the study of proteolytic activity in live, intact cells

  • intracellular protease activity can be visualized using a standard fluorescence microscope, as well as by multi-parameter flow cytometry
  • the substrates contain amino acid sequences from both sides of cleavage sites, providing a degree of specificity not found with substrates made with only amino terminal information

  • the substrates mimic active site loop conformation of proteases and thus provide a three-dimensional target for proteases.

Liliana Guedez, a research fellow at the National Cancer Institute, has found PhiPhiLux particularly useful in getting a handle on the specific pathway inducing apoptosis in her system. In her hands it's fast-being detectable within 15 minutes of the onset of apoptosis; it's more specific than the other early markers of apopotosis; and it doesn't interfere with the process and hence can be added to and followed in live cells.

For more information, visit the Oncoimmunin web site at www.access.digex.net/~oncoim/

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