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aCSF was removed by careful suctioning and replaced with aCSF containing vehicle (aCSF+aCSF), 100 ng/ml with carrier free adult recombinant Hepatocyte Growth Factor (HGF) (R and D Systems, MN, USA) in aCSF, 10 10 M Hinge (Harding lab), 50 ng/ml in aCSF, 10 10 M Dihexa (Harding lab) in aCSF, 10 12 M Dihexa in aCSF or 50 ng/ml HGF+10 12 M Dihexa in aCSF for 30 minutes at 37 C

The 10ml volume is suitable for multiple reconstitutions in standard research laboratory applications

Drug displaced from plasma protein will of course distribute throughout the volume of distribution, so that a 5% increase in the amount of unbound drug in the body produces at most a 5% increase in pharmacologically active unbound drug at the site of action

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The samples were then centrifuged at 2,000 g for 15 min at 4C to separate the serum

doi: 10.1161/CIRCULATIONAHA.116.026099