Imaging was done as described previously (Melcer et al., 2012 (link)). In brief, we used a Revolution spinning disk (CSUX; Yokogawa) imaging system (Andor) mounted on an IX81 fully automated microscope (Olympus) equipped with an automated stage and an environmental chamber (LIS) controlling humidity, CO2, and temperature. FRAP was done using a specialized FRAPPA module (Andor) at 100% 488-nm solid-state laser (50-mW) intensity. We used an EMCCD iXon+ camera (Andor) with a window size of 512 × 512 pixels. H1.0/H1e-GFP recovery was measured over 45–90 s at one or two frames per second. All FRAP experiments were completed within 1 h after addition of KCl into the culture media. The FRAP analyses were performed on 15–30 cells from at least two independent experiments. Two-tailed Student’s t test was performed to compare the kinetics of the different FRAP curves.
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FRAP Imaging of H1.0/H1e-GFP Dynamics
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Corresponding Organization : Gunma University
Other organizations : Kyoto University, Jackson Memorial Hospital, University of Mississippi Medical Center
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Variable analysis
independent variables
- Addition of KCl into the culture media
- H1.0/H1e-GFP recovery measured over 45–90 s
- Imaging system (Revolution spinning disk (CSUX; Yokogawa), IX81 fully automated microscope (Olympus) equipped with an automated stage and an environmental chamber (LIS) controlling humidity, CO2, and temperature)
- FRAP done using a specialized FRAPPA module (Andor) at 100% 488-nm solid-state laser (50-mW) intensity
- EMCCD iXon+ camera (Andor) with a window size of 512 × 512 pixels
- No positive or negative controls were explicitly mentioned in the provided information.
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