for conducting experiments with a bovine hepatocyte cell line, I am looking for a good protocol to establish an EROD assay for measurement of CYP1A1 activity.
Is there anyone who could help me out?
Thank you.
Dear Alexander,
7-Ethoxyresorufin O-deethylation (EROD) is to be carried out in 24-well plates in a volume of 300 μl/well. Incubation mixture: 50 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl2, and 0.5 mM NADPH. For EROD, cell cultures are incubated with 10 μM 7-Ethoxyresorufin for 1 h at 37°C. After 10 min incubation, the reaction is stopped with 1.6 M glycine buffer (pH 10.3). Formation of resorufin and coumarin is quantified by fluorometry in 96-well plates with a fluorescence plate reader. Fluorescence is determined at λEx = 530 nm and λEm = 580 nm for resorufin.
For more details (we used human HepG2 cell line, where vaccinia virus system was used to express recombinant CYP1A1), - please read our paper in Archives of Biochemistry and Biophysics, volume 307, pp. 259-266, 1993.
Best wishes,
Ilya
@Ilya Tsyrlov i need your advice in setting up an EROD assay on HepG2 cell line? I am Having some trouble in performing. As there are different conditions for tissues and cells.
Dear Sir Ilya Tsyrlov
Can you please explain me why it is necessary to add the NADPH in the incubation mixture? I found in some other literature that NADPH is added to start the EROD reaction. I want to know the exact role of NADPH in EROD assay.
Thank you for your time.
http://rsbl.royalsocietypublishing.org/content/roybiolett/suppl/2010/08/18/rsbl.2010.0600.DC1/rsbl20100600supp1.pdf
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