FRET-based evaluation of Bid cleavage in a single primary cultured neuron.

Apoptosis is a cell death modality that is initiated by the activation of caspases. Theoretically, fluorescence resonance energy transfer (FRET) analysis should be a convenient tool for visualizing the activation of caspase. Since the FRET probe cannot be transfected in primary neuronal cultures effectively, the FRET signal is not sufficiently strong for evaluations. We developed a method of extracting the significant signals from the fluorescent FRET images that enables the initiation of apoptosis to be analyzed. We used primary hippocampal cultures transfected with a vector encoding Bid fused with YFP and CFP. Apoptosis was induced using staurosporine (STS; 1μM). The CFP and YFP signals were observed using an inverted fluorescence microscope and were processed using imaging software for analysis. After the background signal was subtracted, the area of caspase activation and the significant signals were extracted from the localized intense signals originating from mitochondria. The CFP and YFP intensities of a selected area in a single neuron were integrated, and the CFP/YFP ratio was obtained. To confirm caspase activation in a similar experimental setting, a luminescence analysis was also performed. The FRET signals from the cultured neuron were confined to foci, since the Bid linker was specifically localized in the mitochondria. The extracted CFP and YFP signals from the foci were strong enough to be evaluated. The average CFP/YFP ratio in the neuron increased significantly after an STS challenge, from 0.673±0.024 (control) to 1.008±0.134 (STS) (mean±SD) (P<0.05). Our study demonstrated, for the first time, the quantification of Bid cleavage as expressed by FRET in a primary neuron. Since Bid is localized in the mitochondria, the region of interest was restricted to a specific area, enabling the signal to be analyzed. This methodology may be useful for the application of FRET analyses in primary cultured cells.