Biology Reference
In-Depth Information
8.
In most cases, the agarose should easily separate from the slice. If the agarose is
still attached, separate it from the slice using a pairs of forceps in each hand.
Cutting the agarose with a scalpel blade may help. Although the slice can be
mounted onto its support with the agarose attached, be aware that the agarose
may detach after time in the culture.
9.
Culture inserts can also be coated.
10.
This may be done with a forceps or by lifting the slice with a flat spatula. To
ensure attachment, use a forceps to lightly press the slice onto the disk in an
unimportant region of the tissue. Slices can be similarly placed into inserts held
under the fluid level.
11.
Holders float in water. The rim of the holder should be on the bottom.
12.
The slice should always remain under the surface of the liquid. Use a forceps to
lightly press the disk down so that it is flush with the rim of the holder.
13.
Surface tension at the air/liquid interface can damage the tissue or remove it from the
disk if the disk is simply pulled from the PBS/glucose after mounting the slice.
14.
This step removes most of the PBS. It also further pushes the slice onto the disk
by the force of the capillary action. Alternatively, the holder and disk can be
placed on parafilm and the PBS removed by careful pipeting. Inserts can be
treated similarly.
15.
Complete medium with DMEM is buffered for an 8% CO
2
atmosphere, but other
medium may be different.
16.
Large tissue chunks should be cut into smaller pieces. Volumes and additions can
be scaled up for large amounts of tissue.
17.
The optimal protease concentration and length of incubation may vary for differ-
ent tissue types.
18.
Rotate the pipet within the flame to get an even taper. The optimal bore size will
depend upon the tissue type. Too small of a bore will kill cells, whereas a hole
too large will not effectively dissociate the tissue. We routinely used bore sizes
of 0.2-0.4 mm for perinatal mouse nervous tissue. Instead of flame-polished
glass, a disposable pipet tip (e.g., 1 mL blue tips) may work as well.
19.
If the entire suspension does not fit into the pipet, divide the suspension
among more than one tube into volumes that fit the trituration pipet , and triturate
each individually.
20.
Repeatedly triturating cells that are already dissociated will lower cell viability.
If further dissociation is necessary, allow the undissociated tissue chunks to settle,
transfer the dissociated cell suspension to another tube, add more medium or
BSS to the chunks, and continue to triturate them. Pool all fractions when done.
A longer protease digestion (
Subheading 3.3.2.
) may promote easier dissocia-
tion during the trituration step, but can also lower cell viability.
21.
The optimum concentration for CellTracker™ dyes will depend upon the cell
type and length of culture and should be empirically determined. The optimal
labeling concentration should not be exceeded because of the toxic effects of
DMSO and high dye concentrations. We used 14
µ
m CMTMR for labeling pri-
mary embryonic neurons.
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