This article describes a method for imaging mouse embryonic primary cerebral cortex cells, focusing on progenitor and neuronal cells. The protocol includes transfection for fluorescent labeling and tracking of cell dynamics over time.
Take a polymer-coated multi-well plate containing mouse embryonic primary cerebral cortex cells, such as progenitor and neuronal cells. Progenitor cells are transfected to express fluorescent proteins for labeling and tracking.
Transfer the plate to the preheated incubation chamber of an inverted fluorescence microscope for a stable imaging environment.
Locate the reference position on the plate for consistent imaging locations.
Select multiple imaging fields per well at higher magnification to maximize the likelihood of observing transfected cells.
At regular intervals, simultaneously capture phase-contrast images to reveal cell morphology and dynamics, and fluorescent images to monitor fluorescent protein expression.
Compare time-lapse phase-contrast and fluorescent images to track progenitor cell division and differentiation into neural lineages.
To image retrovirus-transduced cells, place the tissue culture plate into an imaging chamber connected to temperature and carbon dioxide controllers to maintain the cells at constant conditions of 37 degrees Celsius and 5% carbon dioxide.
Select a position in the XY axis to serve as a zero point, and calibrate the zero point. Select 10 to 15 positions per well to be imaged at 10 times magnification. Set the software to acquire phase contrast images every five minutes and fluorescence images every three hours to decrease phototoxicity. Check and adjust the focus in the first three hours of the experiment, as it may change while the temperature equilibrates.
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