Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector.

Reactive Oxygen Species (ROS) are an important part of the normal cell growth cycle and play essential roles in many biological functions. Many techniques have been developed to detect and measure the amount of ROS present in cells. These techniques include spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis with laser-induced-fluorescence detector (CE-LIF) and capillary electrophoresis-on-a-chip with LIF. As ROS has a short half-life outside of the cell, various fluorescent probes, such as dihydrorhodamine 123 (DHR 123), that are membrane permeable have been used in the detection of intracellular ROS. In this paper, micellar electrokinetic capillary chromatography (MEKC) was coupled with LIF detector. Fluorescent probe, 3′-(p-aminophenyl)-fluorescein (APF), was used to detect specific ROS in Chinese Hamster Ovary (CHO) suspension cells as well as attached mouse bone marrow-derived dendritic cells (BMDCs). Separation buffer composition was optimized at a concentration of 25 mM tetraborate buffer with 50 mM sodium dodecyl sulfate at pH 9.3 and lysis of the cells was done successfully with a buffer of 70% ethanol and 0.1mM sodium dodecyl sulfate using a cell amount of 1 × 107. ROS in cells was successfully analyzed by MEKC-LIF method developed, with acceptable RSD for time at 1.54% and area at 2.81%. [ABSTRACT FROM AUTHOR]