Biology Reference
In-Depth Information
Fig. 2. Schematic representation of filled wells, indicating the level required to
avoid flooding or bubbles. This experiment has been set up to assay the effect of block-
ing antibody on PDGF-stimulated migration. It allows six replicates of each PDGF
concentration in the presence or absence of antibody.
5.
When drying the filter, suspend it from the roof of a horizontal laminar flow hood
by a bulldog clip stuck to the hood with sticky tape. This way the filter is held
secure, and dries quickly without touching anything else. When removing from
rinse before drying, do not blot the filter to speed up the drying, as this may affect
cell migration, either by blocking the pores, or by affecting the homogeneity of
the adhesive coating.
6.
When coating the filter with ECM proteins, place the filter onto a 100-200
ยต
L
drop of protein on a clean microscope slide in the humidified chamber.
7.
The microchemotaxis chamber can be used for several different cell types
(8)
,
including neutrophils
(9)
and leukocytes
(10)
as well as oligodendrocyte precur-
sor cells
(2)
. OPCs are usually obtained from mixed glial cultures, and contami-
nating microglial cells need to be subtracted from the cell population prior to the
assay. Preparation of each cell type to be used will obviously differ.
8.
If the chamber is not quite dry before you use it, aspirate the wells using a yellow
Gilson tip, do not use glass which might scratch the chamber.
9.
When placing your test solution into the wells of the lower plate, it is important
to form a positive meniscus on the top of the well (
see
Fig. 2
). A negative menis-
cus will result in a bubble forming on the underside of the filter. However, it is
important to not have too large a meniscus, otherwise you may get flooding and
cross-contamination from well to well. You will find that each chamber has a
different lower well volume, and this can vary by several microliters. It helps to
have already ascertained the volume for each chamber before you start your first
experiment. The volumes should not change with use of the chambers, however,
your pipets might change, and recalibration periodically might also cause slight
changes. So check carefully each time, to ensure that you are not under- or over-
loading the wells.
10.
When designing your experiment, it is important to remember to have control
and test solutions on each chamber (
see
Fig. 3
). Although differences between
chambers should not occur, it is possible. In order to be able to confidently com-
pare test-against control, it is safest to assume there might be.
11.
Place the filter shiny side down onto the chamber. This is according to the
manufacturer's instruction, and does not appear to make a difference to migra-
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