Biology Reference
In-Depth Information
tion if you get it wrong, but it is as well to be consistent. When you place the filter
on the lower plate, first, place the middle of the filter down and allow the ends to
“drop” into place. It important to not move the filter after the first placing, to
avoid cross-contamination from well to well, or bubble formation on the under-
side of the filter.
12.
The silicon gasket is present to allow formation of a water tight seal between the
upper and lower plates of the chamber. It is important to check the gasket for
tears, dimples, or other damage which might jeopardize formation of a complete
seal, including the little bit of filter you have just cut off the corner.
13.
When assembling the chamber, place the silicon gasket onto the pins, without
pushing it onto the filter. At this point, if the filter lifts off the lower plate, bubbles
might form on its underside. The pins will hold the gasket above the filter until
you push the top plate down onto the bottom plate. At this point, you need to keep
constant, even pressure on the top plate so that the filter does not lift slightly.
14.
Once the chamber is assembled correctly, it is important to place the chamber in an
appropriate CO
2
atmosphere, as alkaline conditions will inhibit cell migration, there-
fore, you need to try to equilibrate the Sato's pH as much as possible. It is also helpful
to humidify the chamber, which makes adding the cells easier (
see
Note 16
).
15.
Oligodendrocytes are inclined to clump together, and a single-cell suspension is
required for migration. Therefore, it is necessary to disaggregate the cells by
trituration prior to plating into the chamber. If you are using a different cell type,
this step may be omitted.
16.
When adding the cells to the upper well, it is vital that there are no bubbles in the
well, which will prevent the cells reaching the filter. There are several ways to do
this, but if the chamber has been humidified first it is easier. If the wells are
already wet, there is less likelihood of bubble formation when adding the cells,
and the small amount of moisture is not sufficient to affect the osmolality of the
medium. It helps considerably if the medium in use has been warmed to 37
C and
swirled to remove bubbles from the tube. This will allow dissolved gases to come
out of the solution before you put it in the well. One way to add the cells is to
place the pipet tip on the surface of the filter, then inject the cells in a quick,
smooth action. However, this method runs a high risk of puncturing or stretching
the filter surface. A better way is to place the pipet tip against the side of the well,
angled (
see
Fig. 4
), injecting the cells in a quick smooth action. If a bubble does
form, which you will know because the cell suspension will not fit into the well,
the size of the bubble will determine how much above the top of the well the
meniscus forms. Simply remove the cell suspension and reinject it, or remove the
bubble with the pipet tip, reinjecting any cells that you remove in this process.
Sometimes very small bubbles form, which barely affect the meniscus. To check
for such bubbles, look directly into the well from above, bubbles can also be seen
though the sides of the chamber. It is important to remove any bubbles before the
experiment starts. It is also important to remember that if you remove the cell
suspension from the well, do not place it back into the stock of cells, because
proteins from the filter or lower well may have contaminated the solution, and
you would thus cross-contaminate all the remaining cells.
°
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