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OncoImmunin, Inc. has designed, synthesized, validated, and patented a new class of cell permeable fluorogenic protease substrates. Unique aspects of these probes are: (1) their ability to cross intact cell membranes enables measurement of intracellular protease activities in live cells, (2) incorporation of amino acid sequence information from both sides of protease cleavage sites. i.e. P and P’ residues, up to a total of 10 amino acid residues, provides complete protease recognition sequences, (3) conformation of substrates’ peptide moieties assume loop or extended beta sheet conformations resulting in substrates retaining protease in vivo specificity and (4) the choice of fluorophores permits simultaneous measurement of multiple proteolytic activities in biologic samples.
 

PhiPhiLux® is OncoImmunin, Inc.’s novel and unique class of fluorogenic apoptosis assay substrates for the detection and measurement of caspase 3 and caspase 3-like activities in living cells (for other apoptosis-specific fluorogenic substrates [CaspaLux® family], see below). The presence of the amino acid sequence of DEVDGI allows one to determine the apoptosis specific, intracellular caspase-3 (CPP32) and caspase-3-like activities by flow cytometry or fluorescence microscopy. By judicious choice of fluorophores, either green or red fluorescence (or under license, far red) may be observed. Cells are neither permeabilized nor fixed. The principle used in the design of these apoptosis reagents was derived from exciton theory. See the PhiPhiLux® review which appeared in The Scientist

Detailed PhiPhiLux® instructions are available: (click icon for download):

PhiPhiLux® G1D2 green fluorescence

PhiPhiLux® G2D2 red fluorescence

The range in sensitivity between a T-Cell leukemic (Jurkat cell, left below) and a Burkitt’s lymphoma (SKW 6.4, right below) line to different apoptogens is exemplified by PhiPhilux®.   Control αFas Ab Staurosporine		SKW6.4 ± αFas Ab + PhiPhilux®- G1D2
		Control	+ αFas Ab

Treatment of a breast tumor cell (231-01) line with TRAIL: Activation of caspase 8 (red = cleavage of CaspaLux® 8)

Give

a try!

cell-permeable fluorogenic substrates for caspases are available in two colors: (click on icon to download protocol) Caspase green red Caspase 1 CaspaLux®-1 - E1D2 CaspaLux®-1 - E2D2 Caspase 6 CaspaLux®-6 - J1D2 CaspaLux®-6 - J2D2 Caspase 8 CaspaLux®-8 - L1D2 CaspaLux®-8 - L2D2 Caspase 9 CaspaLux®-9 - M1D2 CaspaLux®-9 - M2D2

Flow cytometry data from apoptosis studies

No other protease assay is like it!

Specific- peptide sequence is selected to match the desired protease
Sensitive- this is fluorescence, after all!
Broadly applicable - works not only for clear solutions but also cell suspensions

A PhiPhiLux^® kit can be purchased using a Visa/MasterCard credit card by phone, FAX, or e-mail.
Call for special introductory prices.
PhiPhiLux^® is US patented; additional US patent and PCT applications are pending.

With OncoImmunin’s CaspaLux ^® substrates, the activities of caspases-1,6,8 and -9 can be measured. Moreover, by combining the 5 different substrates in complementary colors, multiple caspase activity can be determined simultaneously in live cells by both flow cytometry and microscopy. A high throughput assay is also available.