Liver cells which are exposed to excessive amount of inflammatory mediators (IM) show changes of the mitochondrial function and the endoplasmatic reticulum (ER). It is known, that IM reduce the membrane potential of the mitochondria and also leads to a higher production of mitochondrial reactive oxygen species (mROS). Additionally IM lead to ER stress, which elicits a stress response, the unfolded protein response (UPR). The purpose of UPR is either to restore the ER function, or to prepare the cell for apoptosis.
The hypothesis of this work was that a high production of ROS suppresses the UPR gene expression. The aim of this study was to establish a protocol enabling the sorting IM treated cells in function to their ROS production and mitochondrial membrane potential. From these cells the quality and quantity had to be determined in order to allow a subsequent analysis of gene expression.
In this work adherently growing hepatocytes (line BRL-3a) were treated with media containing IM. The intracellular ROS (iROS) were visualized using the fluorescent dye CM-H2DCF-DA. In order to follow changes in mitochondrial function MitoTracker® Deep Red 633 was used to visualize the mitochondrial membrane potential and MitoTracker® Red CM-H2XRos was applied to determine ROS generation within mitochondria (mROS). The treated cells were analyzed by Laser-Scanning-Microscope and by flow cytrometry following detachment. Treatment of BRL3a cells with IM increased fluorescence of the dyes used for mROS and iROS, which showed a positive correlation. The protocol for fluorescence activated cell sorting (FACS) in function to the iROS and the mitochondrial potential was successfully established and optimized. In order to maintain the differences between treatment and control for the subsequent FACS, the cells had to be stained prior to the detachment. RNA of sufficient quantity and quality (RNA integration number > 7,9) was extracted and can be used for gene expression analysis.