Biology Reference
In-Depth Information
may disintegrate and fragment. Two ways to do this would be to reduce the abso-
lute volume of cell suspension and agarose, at the same time maintaining the
ratio, or by changing the ratio of cells suspension to agarose. Trial and error is
required to do this successfully.
28.
In order to produce a nice round drop, the pipet tip can be lowered to make con-
tact with the plastic at a perpendicular angle and then the 1.5
L volume released.
The substrate for plating the drops should always be dry; if not the 1.5
µ
L tends
to spread further and unevenly, and does not produce an even drop. The number
of drops per dish can be limited, e.g., to 12, to prevent excessive desiccation of
the drops while plating.
µ
29.
It is very easy to dry out the drop excessively during this procedure. This will
manifest an excessive amount of cell death, particularly at the periphery of the
drop. This can be reduced by either cutting down on the time the drops are setting
in the refrigerator (e.g., from 15 min to 7-10 min ) or by plating a smaller number
of drops on each plate (instead of 12 per plate, try only four or six).
30.
Add the medium to the gelled drop by taking up 50
L of Sato's medium into the
pipet tip and then drawing a circle around the agarose drop by slowly releasing
the media. Then, adding the remainder of the 50
µ
L into the center of the circle,
cover the agarose drop. This should be done very carefully in order to avoid
excessive mechanical forces detaching the agarose drop from the plastic dish. It
is also very easy to detach the drop when adding the solution around the drop.
This can be limited by adding the surrounding 50
µ
L of media in a more gentle
fashion, or alternatively, by placing the drop straight onto a PDL substrate (
see
Note 31
). In addition, the plates should be handled with care and not moved
abruptly throughout the experiment.
µ
31.
Because the drops may adhere in the early stages, it is not uncommon for them to
detach during the course of the experiment. This can be avoided by placing the
drops into wells that have been precoated with PDL all over. However, when cell
migration on ECM substrates is to be examined it is advisable not to precoat the
entire surface with PDL because this tends to mask the difference in the effects of
the different ECM molecules. In this case, one approach is to mark the underside
of the well with a fine black point and then add 1
L PDL onto the plastic. This
then evaporates, but acts as additional anchorage for the agarose drop to adhere
to (the black point allows precise positioning of the agarose drop after evapora-
tion of the PDL).
µ
32.
It is essential that the counting procedure remains constant throughout, and
between experiments, you need to ensure that the plates are always aligned in a
uniform manner on the microscope prior to counting the cells.
Acknowledgments
The authors would like to thank Marion Perryman for her technical advice
on the chemotaxis chamber, and Chandike Mallawaarachchi for his helpful
comments on the manuscript. This work has been funded by the Multiple Scle-
rosis Society and the Wellcome Trust.
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