Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

Oxidative stress measurement. The oxidative stress measurement in C. elegans was performed according to a published protocol. 50 Briefly, the ROS was detected in vivo in staged first-day adults C. elegans by staining with 2 0 , 7 0 -dichlorodihydrofluoroscein diacetate (Molecular Probes, Eugene, OR, USA). Nonfluorescent DCFDA is a freely cell permeable dye, which can be readily converted to fluorescent 2 0 , 7 0 -dichlorofluorescein owing to the interaction with intracellular H 2 O 2 . C. elegans samples in triplicate were incubated in 96 well plate containing 50 mM DCFDA in M9 buffer at room temperature (25 1C) in a fluorescent microplate reader (Spectra MAX Gemini, Molecular Devices). C. elegans samples were read every 2 min for 5 h for quantification of fluorescence at 485 nm (excitation) and 530 nm (emission). After the assay, C. elegans samples were harvested to determine protein concentration for normalization.