 |
|
| Dr. Alfiya Bikineyeva |
|
|
|
|
Shimatzu HPLC system |
FRIMCORE is equipped with new state-of-the-art Shimatzu HPLC system with high sensitivity fluorescence detector and temperature controlled (4-40 °C) autosampler. We perform HPLC analysis of cellular and mitochondrial superoxide using dihydroxyethidium (DHE) and mitochondria-targeted mitoSOX fluorescent probes. These probes upon reaction with superoxide produce 2-hydroxyethidium, however, cellular reactions also generate unspecific oxidation products such as ethidium. These products have very similar fluorescent spectra, therefore, editorial panel of Society for Free Radicals in Biology and Medicine have advised to use HPLC over fluorescent microscopy for superoxide measurements in DHE and MitoSOX samples. HPLC analysis of 2-hydroxyethidium provides quantitative and specific analysis of superoxide which can be normalized for cell number, tissue amount or protein content.
We offer superoxide measurements in cell-free, cellular and tissue samples using HPLC, DHE and MitoSOX as following:
1) Measurement of cellular superoxide by DHE/HPLC;
2) Measurement of mitochondrial superoxide by MitoSOX/HPLC.
We recommend scheduling a meeting prior to HPLC service in order to determine the best protocol for sample preparation. This service may require training for sample preparation; however, all samples will be processed by FRIMCORE staff. To schedule a meeting or consultation please contact Dr. Sergey Dikalov or Alfiya Bikineyeva:
- Kalyanaraman B, Darley-Usmar V, Davies KJ, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ 2nd, Ischiropoulos H. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med. 2012;52(1):1-6.
- Fink B., Laude K., McCann L., Doughan A., Harrison D., Dikalov S. (2004) Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroxyethidium and an HPLC-based assay. Am. J. Physiol. Cell Physiology 287(4): C895-902.
- Dikalov S, Griendling KK, Harrison DG. Measurement of reactive oxygen species in cardiovascular studies. Hypertension 2007; 49(4):717-27.
- Dikalova AE, Bikineyeva AT, Budzyn K, Nazarewicz RR, McCann L, Lewis W, Harrison DG, Dikalov SI. Therapeutic targeting of mitochondrial superoxide in hypertension. Circ Res. 2010; 107:106-116.
- Dikalov SI, Harrison DG. Methods for Detection of Mitochondrial and Cellular Reactive Oxygen Species. Antioxid Redox Signal. 2012
|
