The Spectacular Ribonucleotide reductase 'Cheat' That Might Fool Just About All

TTX (1��M) was also added to the culture medium. Fluorescent images of the dendritic shafts were obtained from living neurons using a Zeiss LSM510 confocal microscope, equipped with a stage CO2 incubator (Tokai Hit). Fluorescent resonance energy transfer (FRET) was measured with the donor dequenching after acceptor photobleaching method as described previously (Takemoto-Kimura et?al., 2007). CFP and YFP signals were monitored by independent scans using 458?nm light and 514?nm light as excitation with 470-500?nm (for CFP) and 530-600?nm (for YFP) band-pass filters, respectively. Acceptor photobleaching was achieved by repetitive scans with intense 514?nm laser light on dendritic regions of transfected neurons. Regions of interest (ROIs) for quantification were set on the head of dendritic spines. After background subtraction, Selleck CH5424802 FRET efficiency CFTR activator was calculated as follows: FRETEfficiency(%)=(FCFP, after?FCFP, before)��100FCFP, afterwhere FCFP, before and FCFP, after represent the averaged CFP fluorescent signals before and after the YFP photobleaching, respectively ( Takemoto-Kimura et?al., 2007). In our measurements (Figure?S1C), the FRET efficiency between Arc-CFP and CaMKII��-YFP was relatively low. Nonetheless, the efficiency was significantly higher than the theoretical null value of zero (p?Ribonucleotide reductase for 2?hr and then, just prior to the start of live imaging, the medium was replaced with conditioned medium containing TTX (1?��M) or medium without any inhibitors. Fluorescent images of dendritic shafts were obtained from living neurons using a Zeiss LSM510 laser scanning confocal microscope with excitation at 488?nm for GFP and at 543?nm for RFP. Series of z-stack images for both GFP and RFP signals were acquired in time-lapse mode. The neurons were maintained at 37��C in a stage CO2 incubator (Tokai Hit, Japan) during imaging sessions. Images were quantitatively analyzed using the MetaMorph software. The z-stack images were projected into a single-plane image by summation, and the fluorescence line profiles of well-separated spines and adjacent dendritic shafts were measured. Only the spines that were consistently present during an imaging session were included in the analysis.