KEEP EVERYTHING ON ICE

1.      Cerebral cortex tissues are obtained, dropped into a 50ml tube containing cold culture medium (supplementary info) and kept on ice.

2.      All visible larger blood vessels and the meninges are carefully removed. (Murine brains: also remove all of the ¡°white tissue¡±; brain stem etc.)

3.      Brain specimens are cut into small pieces and homogenized in DMEM-S using a 10 ml Dounce homogenizer with a loose fitting (10 strokes, or until all tissue clumps are removed; ¡°milkshake¡± appearance). 

4.     The homogenate is centrifuged in 15% dextran for 10 min at 10,000g (8000rpm). (Add 30% dextran 1:1 to homogenate eg. If volume of homogenate is 10ml, add 10ml of 30% dextran, add same amount of dextran to ¡°balance¡± tube, weigh tubes before spinning)

5.     Dextran and microvessels will pellet. Remove supernatant including floating ¡°junk¡± and transfer to a new tube to be centrifuged again for 10 min at 10,000g (8000rpm)

6.      Wash pellets from steps 4 & 5 carefully with DMEM-S (?1ml, just roll over pellets & decant). Ensure there is NO junk left in the tube.

7.      Resuspend pellets in DMEM-S in a 15ml tube (5ml), spin @ 900rpm (tabletop centrifuge) for 5min

8.      The pellet containing crude microvessels is further digested in a solution containing 1 mg/ml collagenase/dispase/0.1mg/ml DNase in DMEM-S for 30min-1 hr at 37^oC, until there are no more clumps of pellet. (we used our stocks; working solution was 0.5mg/ml of collagenase & dispase in RPMI, 5ml for 2x murine brains, 30min @ 37^oC on a rotator)

9.  When dealing with very little tissue, it is sometimes better to skip the next step as vessels are ¡°lost¡± due to the absorption process described in step 10. In such cases, the solution from step 8 is plated on the coated dish in culture medium

10.      Microvascular capillaries are isolated by absorption to a column of glass beads and washing off the beads (see supplementary info). Apply microvascular capillaries/enzyme mixture to beads, add RPMI (No serum!!), until the flow through solution goes clear (keep the flow through).

11.      Decant beads/vessels in 50ml tube: use RPMI to ¡°flush¡± the beads out of the 10ml syringe.

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