A New Dual-Promoter System for Cardiomyocyte-Specific Conditional Induction of Apoptosis
Measurement of apoptotic cell death. The amount of apoptotic cell death was determined after 4 hours exposure to 1 μg/mL Tet by using the deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), a standard method for evaluating DNA fragmentation. Following fluorescent labeling, cells were fixed on slides and analyzed by fluorescence microscopy. (a) Cells stained with DAPI (nuclear dye), fluorescein-labeled dUTP, and merging of the two channels. (b) Quantification of TUNEL-positive cells, expressed as the fraction of green fluorescent cells over nonfluorescent cells in each of the 6 fields counted for each condition. Negative control: nontransduced cells; positive control: cells treated with DNaseI; : HL-1 cells transduced with pNRTA vector and nontreated with Tet; +Tet: HL-1 cells transduced with pNRTA vector and treated with Tet for 4 hours.