Biology Reference
In-Depth Information
Fig. 4. Schematic diagram indicating the approximate position and angle of the
pipet tip when injecting cells into wells. Note that the gasket bulges out, this may
cause a bubble to form at the edge of the well.
17.
Cells will require different time periods to achieve adequate migration, e.g.,
microglia will migrate in 4 h
(3)
, as will neutrophils
(9)
, however, 8 h are required
for oligodendrocyte migration
(2)
. Therefore, you need to refer back to the litera-
ture for your cell type or run preliminary experiments to ascertain the time
required by your cells.
18.
When disassembling the chamber prior to staining the filters, take care not to
touch the underside of the filter where your cells are located.
19.
Once fixed in methanol, the filter becomes even more brittle, and has a tendency
to fold or roll up, it is important to avoid the filter surfaces touching each other,
which runs the risk of cells being transferred from place to place, or being scraped
off altogether. This is why two pairs of forceps are useful. The fixing time and
eosin-staining time are not critical, but do not over stain in haemotoxylin, other-
wise, it becomes difficult to distinguish between cytoplasm and nucleus.
20.
Immediately after use, place the chamber in water to avoid the protein solutions
drying out on the filter or gasket.
21.
This is when snipping the corner off of the filter becomes invaluable as a method
of aligning the filters.
22.
Take great care when cleaning the unmigrated cells off the upper surface of the
filter, as it may tear, fold, or crease at this time. The filters are inclined to lift off
the slide and fold over so that you accidentally wipe migrated cells off. It may
help to gently wipe the upper surface of the filter with a wet cotton bud to remove
most of the cells, then to blot the filter carefully, with a paper towel. This helps
the filter stick to the slide better, reducing the risk of it lifting and folding over.
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