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S. J. Budd, A. G. Parker, M. E. Hartnett; NAC Decreases Reactive Oxygen Species Production for in-vitro Choroidal Endothelial Cells Stimulated With VEGF. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3841.
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To determine the influence of n-acetylcysteine (NAC) on the production of Reactive Oxygen Species (ROS) by CECs following VEGF exposure.
Primary human choroidal endothelial cells (CECs) were isolated from donor eyes obtained from the North Carolina Eye Bank. CECs were grown in endothelial growth media (EGM-2) supplemented with 10% FBS. For reactive oxygen species (ROS) production assays, CECs were plated on 96-well plates at a concentration of 3,000 cells per well and allowed to grow in complete growth media (EGM-2) to 80% confluency. Prior to running the ROS assay, cells were pre-treated for 6 hours with EGM-2 media supplemented with 25 mM of n-acetylcysteine (NAC). The ROS production was detected with a 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester (DCF-DA) based assay (Invitrogen, Carlsbad, CA). Cells were incubated with 100 µL of the EBM-2/DCF 5 µM solution for 10 minutes to allow the ROS indicator to permeate the CECs. Following a 30 min incubation with 100 µL of serum free EBM-2 media, cells were incubated for 15 minutes with 10, 50, or 100 ng/ml of VEGF165 to induce ROS production. As a control, cells were incubated with EGM-2 media supplemented with 200 µM hydrogen peroxide to serve as an exogenous source of ROS. Fluorescent measurements were taken on a Wallac 1420 VICTOR2 plate reader using an excitation wavelength of 485 nm and a reading wavelength of 520 nm. Background fluorescence, as determined by cells untreated with the DCF-DA reagent, was subtracted from all data points.
There was an 85% reduction in ROS for cells treated with 25 mM of NAC compared to untreated controls. CECs incubated with 10, 50 and 100 ng/ml of VEGF for 15 minutes displayed a 53%, 61% and 74% increase in ROS, respectively (p<.03). When incubated with VEGF and 25 mM of NAC, the increase in ROS was reduced by 63%, 44%, and 47% (p<.02)
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