116 Slides were incubated at 5.5 or 25 mmol/l glucose at 22 h post-transfection and fixed with formaldehyde 24 h post- transfection for mounting with VectaShield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Each preparation condition was transfected two times. For fluores- cence resonance energy transfer (FRET) studies, cells were seeded at a density of 2×105 cells on 35 mm glass-bottom dishes (MatTek, Ashland, MA, USA) and grown for 24 h. Thereafter, cells were transfected with 1 μg DNA (1:1 ratio of the EYFP and ECFP plasmid) and 2 μl jetPEI (Qbiogene, Montreal, QC, Canada) according to the manufacturer’s instructions. The culture medium was replaced 1 day after transfection, and the cells were incubated for another 24 h with medium containing 5.5 or 25 mmol/l glucose. Hepatocyte isolation, culture, and transient transfection Primary hepatocytes were isolated from C57/BL6 mice using a modification of the collagenase perifusion methods of Seglen [19] and Bader et al. [20]. Isolated cells were suspended in Williams’ medium E supplemented with 10 mmol/l glucose, 5% (vol./vol.) fetal calf serum, 1×10−4 mmol/l dexametha- sone, and 1×104 pmol/l insulin. Hepatocytes were seeded at a density of 4×105 cells on 35 mm collagen-coated glass- bottom dishes (MatTek, Ashland, MA, USA) or on 12-well plates and incubated for 24 h in a humidified atmosphere at 37°C and 5% CO2. Cells were transfected with 1 μg DNA (either EYFP plasmid alone or 1:1 ratio of the EYFP and ECFP plasmid) and 3.2 μl PromoFectine Hepatocyte (Pro- moCell, Heidelberg, Germany) according to the manufac- turer’s instructions. Thereafter, hepatocytes were cultured for 24 h and finally incubated in medium with either 5.5 or 25 mmol/l glucose for 2 h. Fluorescence microscopy and image analysis Confocal images were acquired using a Zeiss LSM 510 NLO Meta system mounted on a Zeiss Axiovert 200M microscope with an oil-immersion Plan-Apochromat 63 × /1.4 DIC objective lens. Excitation wavelengths of 488 nm (2%), 561 nm (5%) and 770 nm (3%) were used for detection of EGFP-GCK, mCherry-GKRP and DAPI, respectively. Confocal images were post-processed using the MediaCy- bernetics Image-Pro Plus v7.0 software package (Bethesda, MD, USA) with a custom macro designed to calibrate, segment (nuclei touching the image border were not included) and outline the nuclei, outline the cell border, count and record measurements. Measurements included nucleus and cell areas (square micrometres) and fluorescent intensity (minimum, maximum, mean, standard deviation and sum). The nuclei outlines were obtained by using DAPI staining as the template and propagating that outline throughout the green and red channels. The cell border outlines were obtained using the green and red signals. Twenty independent transfection control images numbering Diabetologia (2012) 55:114–122 324 total nuclei were processed, along with 143 co- transfection images totalling 1,472 nuclei. To address the possibility that differences in localisation might be driven by differences in fluorescence intensity between variants, cells were also divided into four groups matched for either whole-cell mCherry fluorescence intensity, EGFP fluores- cence intensity or the ratio of mCherry/EGFP intensity in individual cells. For FRET experiments, a cellR/Olympus IX81 inverted microscope system (Olympus, Hamburg, Germany) equipped with a Cellcubator was used, as described by Langer et al. [21]. HC436/24-458BS-HC483/32 and HQ500/24-520BS-D542/27 single-band filter sets were used for ECFP and EYFP, respectively (AHF Analysen- technik, Tübingen, Germany). For FRET analyses, ECFP and EYFP emission were detected simultaneously using a DV-CC Dual View Simultaneous Imaging System (Optical Insights, LLC, Tucson, AZ, USA) equipped with a 505 dcxr beam splitter and D472/30 and HQ542/27 emission filters (AHF Analysentechnik). For FRET analyses and calculation of the ECFP nuclear:cytoplasmic ratio, glass- bottom dishes were fixed on the microscope stage and images were obtained with an UPLSAPO 60 × 1.35 numer- ical aperture oil-immersion objective (Olympus). For calculation of the EYFP nuclear to cytoplasmic ratio on 12-well plates, an UPLSAPO 20× 0.75 numerical aperture objective (Olympus) was used and all motorised devices of the microscope system were synchronised by the scanR acquisition software (Olympus). The image focus was automatically determined with a gradient method from transmitted light images. Finally, the mean fluorescence was calculated as average grey value in selected regions in the nucleus and cytoplasm in individual cells after background correction. The sensitised emission-based FRET efficiency (FRETN) was calculated from the ECFP emission with excitation at 436 nm, EYFP emission with excitation at 436 nm and EYFP emission with excitation at 500 nm, based on the calculation of Vanderklish et al. [22]. FRETN measurements were validated using an ECFP- EYFP fusion construct as described by Langer et al. [21]. Statistical analysis Statistical analyses were performed by ANOVA followed by Bonferroni’s test for multiple com- parison or t tests for independent samples using the Prism analysis program (Graphpad, San Diego, CA, USA). Data are presented as means ± SEM. p values <0.05 (two-tailed) were considered significant. Results Localisation of GKRP fluorescent fusion proteins in HeLa cells and mouse primary hepatocytes To investigate the