Enzymatic dispersal of Cerebral artery SMCs

I.               Vessel dissection and isolation:

    1.     Following deep anesthesia with choice of anesthetics (isoflurane 5%), the mouse is decapitated. Following decapitation, a midline cut is made     to separate the scalp all the way to the nose. Make a cut in between the orbits in parallel to bregma. From the cisterna magna, make a midline cut     on the skull all the way to the previous cut in between the orbits. While making the cut, keep scissors flush with the skull preventing damage to the     brain. Break the cranial nerves without touching the pial arteries on the inferior region of the brain.

    2.     Gently push the brain out of the skull using a blunt tool. Drop the whole brain in ice-cold Mg2+PSS with 6mg/ml bovine serum albumin                 (BSA).

    3.     Dissect the pial arteries off the brain without directly touching the vessels using a spring scissor and fine forceps. Try to clean up the neuronal     tissue and connective tissue by blunt dissection using two fine forceps.

    4.     Transfer the clean arteries to a 1.7ml tube containing Mg2+PSS

II.              Enzymatic digestion of pial arteries to generate single smooth muscle cells.

        1.     First digestion: incubate in 1mg/ml papain (Worthington), 1mg/min dl-dithiothreitol (Sigma), 6mg/ml BSA (Sigma) in 37 C for 12min. Note             BSA makes the solution heavier than the blood vessel, the vessel tends to float. Be careful when transferring the solution.

        2.     Wash the vessels three times with Mg2+PSS.

        3.     Second digestion: incubate in collagenase type-2 (Worthington).

        4.     Wash the vessels three times with Mg2+PSS.

        5.     Put 400-600ul cold Mg2+PSS (for each brain worth of pial arteries) to triturate. Triturate digested segment by gentle suction and release.                 Repeat to break all arteries and release smooth muscle cells. Note: do not incorporate air bubbles which will shear the cells.

        6.     Store cell suspensions generated in step5 on ice until use.

Solutions:

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