Biology Reference
In-Depth Information
3. Procedures
The smooth muscle cells from cerebral arteries of dogs (
1-4, 6, 7
),
rabbits (
5, 8, 9
), rats (
13-15
) and humans (
9
) have been used so
far for studying vasospasm after SAH (Fig.
2
). Smooth muscle cells
usually are isolated by cutting the artery into small pieces and
incubating the pieces for 30 min in enzymes (500 U/ml collage-
nase type IV, 50 U/ml elastase, 100 U/ml DNase I and 1 mg/ml
trypsin inhibitor). Smooth muscle cells are then separated by gentle
trituration while suspended in a buffer solution (dissection solu-
tion + 0.2% bovine serum albumin), stored at 4EC, and used within
12 h of isolation. The identity and health of isolated smooth muscle
cells can be confi rmed by positively stained for
3.1. Dissociation
of Smooth Muscle
Cells from Arteries
-actin (a marker
for smooth muscle cells) and contraction in response to agonists
(KCl, 60 mM or serotonin, 1 mM).
α
Smooth muscle cells are allowed to adhere to a glass coverslip
mounted at the bottom of the recording chamber. Borosilicate
glass pipettes with resistances of 2-5 m
3.2. Whole Cell Patch
Clamp of Smooth
Muscle Cells
are advanced with the
micromanipulator slowly onto the membrane of the cell to be
patched. One then applies gentle suction to the pipette through
tubing attached to the side port of the pipette holder. This forms a
gigaseal with the cell membrane [the seal resistance usually should
be more than 10
9
Ω
(gigaseal)). Next, the cell membrane of the
cell is ruptured by applying a sudden stronger suction to achieve
the whole-cell confi guration.
Ω
Fig. 2. (
a
) Acutely dissociated smooth muscle cells from normal dog basilar artery. (
b
) Normal dog basilar artery smooth
muscle cell with patch pipette advanced onto the cell membrane.
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