Biology Reference
In-Depth Information
camera, and a cell incubation system are required. In our experience, a large envi-
ronmental chamber that encloses the stage and objectives works best. Although it
takes longer to equilibrate temperature, humidity, and CO
2
levels compared to smal-
ler alternatives, temperature control and stability of the optics can be maintained
more precisely. Alternatively, mini-incubators such as those from Tokai-Hit can be
used, but temperature is less well controlled, and the incubator must be disassembled
and reequilibrated every time samples are changed.
Although any fluorescence microscope can potentially be used for these exper-
iments, we prefer wide field systems over scanning laser confocal microscopes
because we encounter less severe problems associated with photobleaching and
photodamage to the cells. To minimize exposure to the UV light source, it is imper-
ative to use an automatic shutter that only opens during image capture and to equip
the microscope with a sensitive digital camera that minimizes exposure times. In
addition, it is useful to have a mechanical stage for the precise placement of cells
into the field of view and software that allows on-screen placement and focus of
cells using short-exposure image capture. We have tested a number of systems for
this work and favor the solutions provided by Deltavision (Applied Precision,
Issaquah, WA). Typically, we use an Olympus 100
objective with a numerical
aperture of 1.35.
4.4.2
Cell preparation
We typically plate cells into 35-mm dishes at a density that will grow to 50-60%
of confluence after an overnight incubation. EGFP-MAP4 plasmid DNA is then
transfected, and the cells are again grown overnight, or until the cell density is about
70-80% of confluence. This density usually gives well spread and healthy cells for
imaging. If the cell density is too high, the cells should be subcultured and grown to
the proper density before continuing. For the high power objectives required in these
studies, glass-bottom dishes such as those from glass-bottom-dishes.com will be
needed. Alternatively, we have found that primary cells such as human vascular
endothelial cells attach much better to plastic. In these cases, plastic dishes of
sufficient optical quality (e.g.,
-dishes from Ibidi LLC, Verona, WI) are used
(
Ganguly, Zhang, Sharma, Parsons, & Patel, 2012
). Once the cells are ready for im-
aging, the medium is replaced with fresh medium containing 25 mM HEPES to help
maintain the proper pH. Although this medium may contain phenol red, better con-
trast is usually obtained in dye-free medium. Note that the use of HEPES-containing
medium is not necessary if the stage incubator is equipped with CO
2
control.
m
4.4.3
Data collection
Before collecting images, we scan the dish with a low power objective to find regions
containing a high number of cells with GFP-labeled microtubules, low background
fluorescence, and a clearly discernable centrosome on the ventral surface as shown in
Fig. 4.1
A. We then switch to a 100
objective and focus on the centrosome by
