Hello to all, the oxidative burst assay which is based on intracellular oxidation of DHR123 does NOT analyze mitochondrial H2O2. It rather indicates any H2O2 production which intracellularly occurs in cells which do express peroxidases. Peroxidase activity is needed for H2O2 dependent oxidation of DHR123 similar to the mechanism of oxidation of DCFH. The major difference between the two methods is that R123 as the product of the oxidation of DHR123 is more efficiently trapped intracellularly than DCF through its high affinity to mitochondrial binding sites. Mitochondria are, however, neither the site of the oxidation of DHR123 nor is their activity limiting for the level of fluorescence achieved. Concerning question 2, many researchers in deed have used confocal microscopy to locate intracellular sites of oxidation. Confocal microscopy, however, based on the confocal principle to analyze only fluorescence from a single focal plane within a cell is not quantitative regarding the total level of cellular generation of the fluorescent products. Gregor Rothe > Date: Wed, 19 May 1999 18:52:07 -0500 (CDT) > From: SHAILAJA KALYANKRISHNA <SKALYANKRISH@utmem1.utmem.edu> > Subject: WHAT CONC. OF DCFDA FOR FLOW CYT.?? > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Importance: normal > Priority: normal > > Hello flow cyt. users, > > I intend to measure intracellular H2O2 in vascular smooth muscle cells using > H2DCFDA by flow cytometry. > > QUESTION 1: > > Does anyone know what conc. (nM or microM) of DCFDA the cells should be loaded > with? I am not fixing the cells and I am carrying them to the flow cytometry > facility (10 minutes walk), so I expect some leakage of the dye from the cells. > > I read that DHR123 is more sensitive, but I wish to measure net intracellular > H2O2, rather than mitochondrial H2O2, hence the choice of H2DCFDA. > > QUESTION 2: > > Some researchers use fluorescence confocal microscopy for the same purpose? > Are confocal and flow cyt. equally sensitive and accurate for this purpose? > > Thanks in advance, > Kalyankrishna S. > SKALYANKRISH@UTMEM1.UTMEM.EDU > > *********************************************************** Institute for Clinical Chemistry and Laboratory Medicine University of Regensburg Franz-Josef-Strauss-Allee 11 D-93053 Regensburg, Germany Tel. +49 (941) 944-6204 Fax +49 (941) 944-6202 E-mail: Gregor.Rothe@KLINIK.UNI-REGENSBURG.DE ***********************************************************
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