Live Cell Fluorescent Reactive Oxygen Species Detection Assay

In healthy aerobic cells, reactive oxygen species (ROS) generation typically occurs at a controlled rate. But under stress conditions or by application of various drug candidates, ROS production can be greatly increased.  The resulting changes in many cell components including DNA, proteins and lipids can influence cell viability, metabolism and growth. We have developed a live cell, high-throughput, microtiterplate-based ROS assay (M1049) that utilizes the cell permeable substrate, 2',7'-dichlorofluorescin diacetate, a reliable fluorogenic marker for ROS detection. Upon enzyme activity, the highly fluorescent dye, 2',7'-dichlorofluorescein is produced, with EX: 495nm and EM: 530 nm. By using such a diacetate derivative, the assay can be efficiently adapted for intracellular applications, since the acetates help with effective loading into live cells.  The acetates are then cleaved by endogenous intracellular esterases inside the cell, releasing the intact substrate. Application of this assay to the kinetic and end-point measurement of ROS activity ...more

New Red Bioluminescent Luciferase Vectors

Researchers at Marker Gene have developed a new luciferase gene using point mutation and codon optimization of the luciferase sequence from the natural luciferase gene isolated from Luciola cruciata (the Japanese firefly). This novel recombinant DNA has been incorporated into vectors (M1394 and M1395) containing the highly expressing CMV and SV40 promoters for high expression activities in mammalian cells. The gene codes for an improved amino acid sequence which exhibits long-wavelength light emission (red color, EM 619 nm), as well as improved thermostability and higher expression levels in mammalian cells compared with other luciferases including the native luciferase derived from the American firefly Photinus pyralis. The expressed enzyme uses the same substrate, D-Luciferin (M0237), that is used with other luciferases, and activity can easily be detected using our convenient MarkerGeneTM Live Cell Luciferase Assay Kit (M0626). Additional substrates for use in detecting cloned activity in living tissues and in vivo include the membrane permeant D-Luciferin, ethyl ester (M0906). This makes these new vectors ideal candidates for use in vivo detection methods ... more

MarkerGene^TM Cellular Senescence Microtiterplate Assay Kit

Studies have shown that ß-galactosidase histochemical staining at pH 6 is a useful enzymatic marker of cellular senescence (the so-called senescence associated beta-galactosidase; SA-ß-Gal). While cells entering senescence show this enzymatic activity, immortalized cell types, including tumor or other quiescent cells, are not stained under these conditions. The implications of these methods in such diverse areas as age-related pathology research, tumor analysis and tissue culture maintenance is significant. The SA-ß-Gal assay is one of the most widely accepted markers of cell aging. Often analysis is performed using the chromogenic substrate X-Gal. However, the substrate X-Gal has limited sensitivity and is difficult to quantify. The degree of blue staining in positive cells varies both with cell type and staining conditions, and this introduces subjectivity to results. The MarkerGene^TM Cellular Senescence Microtiterplate Assay Kit (M1405) uses the high-sensitivity substrate fluorescein di-ß-D-galactopyranoside (FDG) to quantify senescent cells. FDG turnover produces the highly fluorescent product fluorescein, which is easily detected using an appropriate filter set for fluorescein fluorescence (i.e. EX 485nm and EM 535 nm) in a microtiterplate assay format. This assay ... more