- Cellular Luminescence Enhancement System for Superoxide Detection
- Easy to Use - Specific for Superoxide
- From 100-Fold to 600-Fold Enhancement
- Requires Fewer Cells - Non Cytotoxic
Diogenes Cellular Luminescence Enhancement System is a superoxide chemiluminescent enhancer that is non-denaturing to living cells. Superoxide radical (O2-) is produced intracellularly as a consequence of aerobic metabolism and extracellularly by leukocytes in response to infection. The extent of oxidative burst produced by white blood cells (WBCs) when stimulated by f-met-leu-phe, phorbol esters, anti-Fc receptor antibodies or LPS is a partial indicator of the immunocompetence of the cells tested.
Currently, the production of O2- by leukocytes is monitored by such cumbersome and indirect methods as measuring oxygen uptake in a Clark electrode (both in the presence and absence of cyanide) or measuring spectral changes caused by the reduction of cytochrome c. As a non-cytotoxic intermediate in the mechanism of photon production, Diogenes is ideally suited to the detection of cell-mediated superoxide production. The intensity of light produced by Diogenes in the presence of superoxide is directly proportional to the O2- concentration, but is much higher than that achieved by using luminol. Therefore, Diogenes is ideal for monitoring cellular immunocompetence, utilizing a luminometer to quantify the light output. Any stimulant that activates an oxidase to produce extracellular superoxide is usable with Diogenes. Such means can be physiological ormimetic of the physiologic pathway.
Diogenes Protocol
Solution Preparation
1. Add 1.0 ml of deionized water to the Diogenes Reagent that is
contained in the vial. Mix until completely dissolved.
2. Add 9.0 ml of deionized water to the Diogenes Activator that is
contained in the bottle. Mix until completely dissolved.
3. Add the contents in the vial (Diogenes Reagent) to the contents in
the bottle (Diogenes Activator), recap the bottle and shake vigorously
to mix.
4. The combined solution (10 ml) comprises the Diogenes Complete
Enhancer in its ready-to-use, final working strength, and will yield
up to 100 assays.
Assay
1. Add 5X104 - 5X105 cells contained in glucose media into a luminometer
cuvette or microtiter plate.
2. Add 100 μl of Diogenes Complete Enhancer Solution.
3. Add 20 μl of stimulant (PMA, mAb197, fMLP, etc.).
4. Read the results. Time to peak output will depend on the cell and
stimulant used. The response of Diogenes to superoxide is instantaneous.