Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.
Total time
~4–6 hours (transfection to imaging) plus 24–48 hrs for cell expression
Model organism
HUVEC (Human Umbilical Vein Endothelial Cells)
Steps
1
Transfect HUVEC with GFP-actin plasmid
Introduce GFP-actin expression vector into cultured human umbilical vein endothelial cells using standard transfection protocols. Allow sufficient time for GFP-actin expression before imaging.
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2
Assemble live-cell imaging chamber and stage heater
Prepare the microscope stage with a temperature-controlled chamber and heating system to maintain physiological conditions (37°C) during long-term imaging of live cells.
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3
Acquire time-lapse fluorescence microscopy data
Perform live-cell imaging using appropriate laser excitation and detection settings to capture GFP-actin dynamics at defined temporal intervals before, during, and after stimulus application.
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4
Generate and analyze kymographs of actin filaments
Construct kymographs from time-lapse sequences to visualize and quantify the spatiotemporal dynamics of actin structures, enabling measurement of polymerization and depolymerization rates.
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