Cyclic AMP was assessed using FRET-based assays. Cells were transiently transfected with the FRET-based biosensors, Epac1-CFP/YFP54 (link) for measuring cAMP and PTHR-CFP with βarr2-YFP for measuring arrestin recruitment. Measurements were performed and analyzed as previously described41 (link). In brief, cells plated on poly-D-lysine coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies), maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl and 0.1–10 mM CaCl2, 0.1% BSA, pH 7.4, and transferred on the Nikon Ti-E equipped with an oil immersion 40X N.A 1.30 Plan Apo objective and a moving stage (Nikon Corporation). CFP and YFP were excited using a mercury lamp. Fluorescence emissions were filtered using a 480 ± 20 nm (CFP) and 535 ± 15 nm (YFP) filter set and collected simultaneously with a LUCAS EMCCD camera (Andor Technology) using a DualView 2 (Photometrics) with a beam splitter dichroic long pass of 505 nm. Fluorescence data were extracted from single cell using Nikon Element Software (Nikon Corporation). The FRET ratio for single cells was calculated and corrected as previously described41 (link). Individual cells were perfused with buffer or with the ligand for the time indicated by the horizontal bar.
Lucas emccd camera
Manufactured by Oxford Instruments
Sourced in Ireland, United Kingdom
The LUCAS EMCCD camera is a high-performance scientific imaging device designed for low-light applications. It utilizes electron-multiplying charge-coupled device (EMCCD) technology to provide high quantum efficiency and signal-to-noise ratio. The camera is capable of capturing images with high spatial and temporal resolution, making it suitable for a variety of scientific and research applications.
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Lab products found in correlation
4 protocols using lucas emccd camera
1
FRET-Based Assays for cAMP and Arrestin
2
FRET-Based Assays for cAMP and Arrestin
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Cyclic AMP was assessed using FRET-based assays. Cells were transiently transfected with the FRET-based biosensors, Epac1-CFP/YFP54 (link) for measuring cAMP and PTHR-CFP with βarr2-YFP for measuring arrestin recruitment. Measurements were performed and analyzed as previously described41 (link). In brief, cells plated on poly-D-lysine coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies), maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl and 0.1–10 mM CaCl2, 0.1% BSA, pH 7.4, and transferred on the Nikon Ti-E equipped with an oil immersion 40X N.A 1.30 Plan Apo objective and a moving stage (Nikon Corporation). CFP and YFP were excited using a mercury lamp. Fluorescence emissions were filtered using a 480 ± 20 nm (CFP) and 535 ± 15 nm (YFP) filter set and collected simultaneously with a LUCAS EMCCD camera (Andor Technology) using a DualView 2 (Photometrics) with a beam splitter dichroic long pass of 505 nm. Fluorescence data were extracted from single cell using Nikon Element Software (Nikon Corporation). The FRET ratio for single cells was calculated and corrected as previously described41 (link). Individual cells were perfused with buffer or with the ligand for the time indicated by the horizontal bar.
3
Comet Assay for Embryonic DNA Damage
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DNA strand break measurements by the alkaline comet assay were performed on 24 hpf embryos. Pools of five dechorionated embryos per replicate were digested with 1 mg.mL -1 of phosphate-buffered saline 1X/collagenase IV from Clostridium histolyticum (PBS 1 X, 137 mM NaCl • KCl 2.7 mM • Na2HPO4 10 mM • KH2PO4 1.8 mM, pH 7.4, Sigma-Aldrich, Germany)
during 45 minutes at room temperature. Cell suspension was filtered through a 48 µm gauze in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, cells pellet was resuspended in 30 µl of PBS. Then, the comet assay was performed as described by Akcha et al. (2003) . For each cell sample, two slides were prepared. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxicity was assessed by measuring the DNA percentage in the comet tail for at least 50 nuclei per slide.
during 45 minutes at room temperature. Cell suspension was filtered through a 48 µm gauze in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, cells pellet was resuspended in 30 µl of PBS. Then, the comet assay was performed as described by Akcha et al. (2003) . For each cell sample, two slides were prepared. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxicity was assessed by measuring the DNA percentage in the comet tail for at least 50 nuclei per slide.
4
Comet Assay for DNA Strand Breaks in Zebrafish Embryos
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DNA strand break measurements by the alkaline comet assay were performed on 24 hpf embryos. Pools of five dechorionated embryos for four samples per concentration were digested with 1 mg.mL -1 of phosphate-buffered saline 1X/collagenase IV from Clostridium histolyticum (PBS 1X, 137 mM NaCl • KCl 2.7 mM • Na 2 HPO 4 10 mM • KH 2 PO 4 1.8 mM, pH 7.4, Sigma-Aldrich, Germany) solution during 45 minutes at room temperature. Cell suspension was filtered through 48 µm gauze into a 1.5 mL reaction tube in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, the cells pellet was resuspended with 30 µl of PBS. The viability of the dissociated embryo cells was evaluated by a Trypan blue exclusion test. Then, the comet assay was performed as described by Akcha et al. (2003) . Two slides were prepared per sample. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxic assessment was assessed by measuring the DNA percentage in the comet tail (50 cells per slide).
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