Capillary electrophoresis analysis of hydrogen peroxide induced apoptosis in PC12 cells.
Apoptosis is a highly regulated and controlled process of cell death that plays a fundamental role in many biological processes. Abnormal apoptosis of cells is closely related to some diseases such as cancer. Development of a simple and effective method to detect apoptosis is of great importance. In the present paper, capillary electrophoresis (CE) method was applied to distinguish cell apoptosis and necrosis of rat pheochromocytoma (PC12) cells treated by hydrogen peroxide by characterizing the DNA fragmentation. Firstly, effects of separation conditions (voltage, polymer concentration, temperature and injection time) on DNA separation were studied using 100 bp DNA ladders as the analyte. Under optimal separation condition (polyacrylamide coated capillaries: 57.5 cm x 75 microm i.d., effective length: 50 cm; running buffer: 1x TBE containing 2% PVP and 1.2% HEC; separation voltage: 5 kV; temperature: 25 degrees C; electrokinetic injection: 10 kV x 10 s), CE was used to monitor the progress of hydrogen peroxide induced apoptosis of PC12 cells by analyze DNA fragmentation. It was found that normal, apoptotic and neurotic cells had distinct DNA fragmentation patterns analyzed by CE. The results by CE were tested by other current methods (DAPI nuclei staining, flow cytometry analysis and TUNEL) to detect apoptosis and correlated well with those methods. Results show that CE can distinguish cell apoptosis and necrosis, quantify the degree of apoptosis in cells and have advantages of high efficiency, fast sample analysis speed, minute sample consumption and reliable results, which provides an accessorial method in the research of multiple diseases with abnormal apoptosis such as cancer and neurodegenerative diseases.
Minjie
Xie
X
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China. xiemj2000@yahoo.com.cn
Wei
Wang
W
Zhou
Zhu
Z
Yongfei
Yu
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000302:isolation & purification; D019075:Electrophoresis, Capillary; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents; D007211:Indoles; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D016877:Oxidants; C007293:DAPI; D006861:Hydrogen Peroxide