Adult mouse DRG neurons transduced with AAV‐PHP.S (Chan et al, 2017 (link)) encoding HA‐Dendra2‐nucleolin (full length and ΔGAR), or N2a or HEK‐293 cells transfected with HA‐Dendra2‐nucleolin (full length and ΔGAR) expression plasmids were live imaged with the Nikon Ti‐LAPP illumination system equipped with a temperature‐controlled chamber and an Andor EMCCD camera. Cells were incubated with 10 µM Hoechst 33342 in medium for 10 min prior acquisition. Images were acquired in DAPI, GFP, TRITC, and DIC channels at ×60 magnification with Nikon CFI plan apochromat 60× oil lambda objective. Medium was removed, and the cells were immediately refocused and imaged or the t = 0 image, followed by careful addition of 1 ml ddH2O by pipetting and image acquisition using a same setting with 42 s interval for 5 min. Images were analyzed by NIS‐Elements software (Nikon).
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Visualizing Nucleolar Dynamics in Neurons
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Other organizations : Weizmann Institute of Science, University of South Carolina, Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, University of California, San Francisco, University of California, Los Angeles
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independent variables
- Transduction of adult mouse DRG neurons with AAV-PHP.S encoding HA-Dendra2-nucleolin (full length and ΔGAR)
- Transfection of N2a or HEK-293 cells with HA-Dendra2-nucleolin (full length and ΔGAR) expression plasmids
- Live cell imaging of transduced DRG neurons and transfected N2a or HEK-293 cells
- Nikon Ti-LAPP illumination system equipped with a temperature-controlled chamber and an Andor EMCCD camera
- Incubation of cells with 10 µM Hoechst 33342 in medium for 10 min prior to acquisition
- Image acquisition in DAPI, GFP, TRITC, and DIC channels at ×60 magnification with Nikon CFI plan apochromat 60× oil lambda objective
- Removal of medium and immediate refocusing and imaging at t = 0, followed by careful addition of 1 ml ddH2O by pipetting and image acquisition using the same settings with 42 s interval for 5 min
- Image analysis using NIS-Elements software (Nikon)
- Negative control: Cells not transduced or transfected (not explicitly mentioned)
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