JoVE Encyclopedia of Experiments
Neuroscience
0 views • 2:33 min • June 17th, 2025
Take a polymer-coated coverslip containing a neuron-astrocyte mixed culture in the recording chamber.
These transfected cells express calcium-sensitive proteins on the plasma and ER membranes, fluorescing upon calcium binding to track subcellular calcium dynamics.
Add media containing calcium ions and cover the chamber to prevent evaporation.
Place the chamber under a fluorescence microscope.
Calcium ions bind to calcium-sensitive proteins. Upon irradiation with blue light the calcium-sensitive proteins on the ER outer membrane fluoresce green. Use this green fluorescence to locate astrocytes.
Next, irradiate with green light. The calcium-sensitive proteins on the plasma membrane fluoresce red. Confirm the presence of red fluorescence in the same astrocyte exhibiting green fluorescence.
Record time-lapse images of calcium-sensitive proteins on the plasma membrane, followed by the calcium-sensitive proteins on the ER outer membrane.
Analyze the images to observe compartment-specific spontaneous calcium activity in the selected astrocyte.
Mount the coverslip containing the cells transfected with Lck-RCaMP2 and OER-GCaMP6f into the recording chamber of the microscope. Add 400 microliters of
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