Biology Reference
In-Depth Information
Fig. 5. Photomicrograph of migrated oligodendrocyte precursors on the underside
of a filter. A pore in the filter is indicated by the arrow.
23.
When counting the cells, count the nuclei only, as some cytoplasm might appear
through the pores without the cells migrating through (
see
Fig. 5
). One method of
counting is to use a square graticule, count three or five fields of view per well,
depending on magnification (e.g., three fields is adequate at 10
×
, but five fields
are required for 20 or 40
). Your magnification factor will depend on how many
cells have migrated, as you will find it impossible to count the cells accurately if
you are looking at 600-800 per field of view at 10
×
. Whereas if you have only 50
cells migrating through your filter, you can count at a lower magnification. As
long as you count at the same magnification across each filter, and as long as
each filter contains a control against which you compare the results, you can
normalize the data and compare across filters/experiments (
see
also
Note 10
).
24. Migration can also be quantified using a spectrophotometric method, based
on the extraction of stain from the nuclei of migrated cells (for further details,
see
ref.
11
).
×
25.
The agarose solution will solidify if taken out of the water bath for any prolonged
period of time (>1 min depending on the volume), but can be reliquefied on sub-
sequent days for further experiments by reheating in the microwave oven, or at
65
C in a water bath for 30 min. One preparation can last approximately 1 mo.
There is no need to filter either the PBS or the agarose solution because the heat-
ing process sterilizes the solution before every experiment.
°
26.
Transfer the cells to a clean tube to obtain a precise volume and avoid drops of
solution on the side of the tube running down and adding to the 40
L volume,
thus further diluting the agarose drop, which may prevent it from gelling properly.
µ
27.
It is important to optimize the cell density for a particular population of cells; if
the density is too low, cells will not survive, and if it's too high, the agarose drop
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