Live-cell imaging and motility analysis

3 × 10⁵ hTERT_RPE1 cells were seeded in six-well plates over-night before live-microscopy analysis. Then, cells were stained with NucBlue Live Cell Reagent (Thermo Fischer) and imaged every 10 min for 18 h using a humidity- and temperature-controlled inverted wide-field microscope within an environmental chamber (ScanR, Olympus). Nuclear tracking and cell motility analysis were performed as described here^{20 (link)}. For this purpose, specific software in C++ with the OpenCV [http://opencv.willowgarage.com/wiki/] and the GSL [http://www.gnu.org/software/gsl/] libraries were developed. The migration analysis was performed by the C++ software coupled with R [www.R-project.org].

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Poli A., Pennacchio F.A., Ghisleni A., di Gennaro M., Lecacheur M., Nastały P., Crestani M., Pramotton F.M., Iannelli F., Beznusenko G., Mironov A.A., Panzetta V., Fusco S., Sheth B., Poulikakos D., Ferrari A., Gauthier N., Netti P.A., Divecha N, & Maiuri P. (2023). PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1. Nature Communications, 14, 1432.

Publication 2023

NucBlue Live Cell Reagent ScanR

Cell Cell motility Humidity Microscope

Corresponding Organization : University of Naples Federico II

Other organizations : Gdańsk Medical University, Swiss Federal Laboratories for Materials Science and Technology, University of Molise, University of Southampton, ETH Zurich

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Variable analysis

independent variables

Number of hTERT_RPE1 cells seeded

dependent variables

Cell motility
Nuclear tracking

control variables

Humidity and temperature control
Staining with NucBlue Live Cell Reagent

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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