Time-Lapse Imaging of Transfected Primary Mouse Cerebral Cortex Cells

Samenvatting

Source: Landeira, B. S.,et al., Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System. J. Vis. Exp. (2017)

The video demonstrates the use of time-lapse imaging to track fluorescently labeled cortical progenitors, monitor their division and differentiation, and study lineage development in primary cerebral cortex cells

Protocol

1. Time-lapse Video-microscopy

NOTE: This step requires an inverted fluorescence microscope with an incubation chamber (see Table of Materials).

1. After the retrovirus infection, place the tissue culture plate into an incubator connected to temperature and CO₂ controllers.
   NOTE: Preheating the chamber for 3 h prior to starting imaging could help minimize drift and defocus during the acquisition.
   NOTE: The chamber should maintain the cells at constant conditions of 37 °C and 5% CO₂.
2. Select a position in the XY axis to serve as a zero-point (XY = 0). This will allow a reference to find the positions again in the future.
   NOTE: It is advisable to draw a sign on the plate that can be used as a reference to determine the zero point.
3. Select the positions to be imaged at 10X magnification.
   NOTE: Select 10-15 positions per well to increase the likelihood of observing retrovirally-transfected cells.
   NOTE: Long-distance high magnification objectives (20X or 40X) can be used to gain higher-resolution images. Depending on the working distance of the objectives, glass-bottom plates may be required.
4. Acquire phase contrast images every 5 min and fluorescence images every 3 h to decrease phototoxicity. Check and adjust the focus in the first 3 h of the experiment, as it may change while the temperature equilibrates. Check and adjust the focus daily.
   NOTE: We observe that the focus becomes stable after the first 24 h. However, it is advisable to monitor the focus. Alternatively, use a software-based autofocus system.
5. After 7 days, stop the acquisition.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
ImageJ	NIH
tTt	ETH Zurich
Cell observer microscope	Zeiss
The Tracking Tool (tTt) software		https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html