Biology Reference
In-Depth Information
Mowiol mounting medium:
Add 12 ml of 50% glycerol to 2.4 g of Mowiol 4-88 (Calbiochem, 475904).
Mix for 2 h at room temperature. Add 12 ml of 200 mM Tris-HCl, pH 8.5.
Incubate at 50
C (occasionally mixing) until dissolution (
3 h). Pass through
20
C.
a 0.45-
m
m filter, aliquot, and store at
CONCLUSION
This method for making micropatterns using deep UV light is straightforward to
use and does not demand any extravagant instruments. Here, we presented an over-
view of how to make the micropatterns. Further discussion on troubleshooting
and variation of the technique has been published elsewhere (
Azioune, Carpi,
Tseng, Th
ยด
ry, & Piel, 2010
).
Cells on a uniformly protein-coated coverslip are seldom isolated from their neigh-
bors, and often even spread on top of each other, despite seeding reasonably few
cells. They adopt a large variation of shapes and are not easily comparable. Forcing a
predefined shape and size on cells using micropatterning techniques, on the other hand,
has shown to be an efficient way of limiting the number of unknown parameters. Size
and shape can be varied rigorously and independently, allowing study of how they
influence the cellular organization. Since the cells are normalized, analysis can be
standardized and even automated (
Chevrollier et al., 2012, Schauer et al., 2010
), which
would be possible only with difficulty using cells on a conventional substrate. Our
current technique confines cells to 2D organization. Future development using 3D
micropatterning would be a step further in the direction of recreating the conditions a
cell experience in its natural environment (
Ochsner, Textor, Vogel, & Smith, 2010
).
The current method enables studies of how the shape of the cell and the mechan-
ical forces due to spatially well-defined interactions with the substrate influence the
mitochondria and the cytoskeleton. Cells shrink when dying, but there is also a causal
link in the opposite direction, that is, cells die when artificially shrunk by adhesion to
too small patterns (
Chen et al., 1997
). From a physiological point of view, this re-
sponse (cell growth or death) to mechanical deformation of the cell or nucleus is
probably important during development and tissue homeostasis, and its deregulation
might be implicated in developmental defects and cancer. The mitochondria being a
crucial factor in apoptosis (
Hoppins & Nunnari, 2012
), the method described here
should be helpful for better elucidating the mechanisms involved in this process,
and the importance of their fragmentation during apoptosis (
Frank et al., 2001
).
Signaling between cells has been well studied, but less is known of the interactions
between cells when it comes to intercellular trafficking (
Rustom, Saffrich, Markovic,
Walther, & Gerdes, 2004
), although there is evidence of transfer of mitochondria or
at least their DNA between cells (
Spees , Olson, Whitney, & Prockop, 2006
). Using
cleverly designed micropatterns, on which the cells would contact each other in pre-
defined and controlled ways, should greatly facilitate the study of this phenomenon.
