Assessment of Intracellular Calcium in Cerebral Artery Myocytes - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 533

Fig. 3. Freshly isolated native cerebral artery myocytes. ( a ) Cerebral arteries were
enzymatically dissociated to obtain isolated vascular smooth muscle cells. Image shows
individual smooth muscle cells using differential interference contrast microscopy (x40).
( b ) Vascular smooth muscle can be identifi ed by spindle-shaped cell morphology as well
as immunostaining of smooth muscle alpha actin and smooth muscle myosin heavy chain.
Scale bars represent 10 μ m. From Nystoriak et al. ( 28 ) with permission.

80 L -glutamic acid (mono-sodium salt), 2.0 MgCl 2 , 2 CaCl 2 , 10
HEPES, and 10 glucose (pH 7.3; 37°C; 30 min) using a 1 ml vial
placed in a water bath. Arteries are then transferred to a Ca 2+ -free
GIS containing papein (0.3 mg/ml) and 1,4-dithioerythriol
(0.7 mg/ml; 37°C; 17 min) using a fi re-polished Pasteur pipette.
Next, arteries are incubated in GIS containing collagenase type F
(0.7 mg/ml), collagenase type H (0.3 mg/ml), and 100

M CaCl 2
(37°C; 20 min). Finally, arteries are incubated in GIS containing
2 mM CaCl 2 on ice (3×; 10 min) and gently triturated into indi-
vidual cerebral artery myocytes using a small bore fi re-polished
Pasteur pipette. Cerebral artery myocytes can easily be identifi ed
by characteristic spindle-shaped morphology (Fig. 3 ). The cells are
kept on ice and used within 6 h of isolation.

μ