Reaktion #2507859

Reaktionsgleichung

NaCl

citrate

MgCl2

glucose

HEPES

dextrose

KCl

Fura 2/AM

DCFH-DA

→

quercetin

Lösungsmittel

methanol

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeDrug-free human blood obtained from healthy volunteers
2. 2
   WaschenThe platelets were washed by exclusion chromatography on Sepharose 2B
3. 3
   Einengenat concentrations<0.5%

Vorschrift

Drug-free human blood obtained from healthy volunteers was coagulated with acid:citrate:dextrose. Platelet-rich plasma was centrifuged at 800×g for 20 min at room temperature and the pellet was suspended in a volume equal to half the initial volume of autologous plasma, low in platelets. The platelet suspensions were incubated for 1 h at 37° C. with 3 μmol of Fura 2/AM per L, 40 μmol DCFH-DA/L, 7.4 GBq (2 Ci) 32Pi/L, or 3.7 MBq (1 mCi) [3H]oleic acid/L. The platelets were washed by exclusion chromatography on Sepharose 2B using a Ca2+-free Tyrodes buffer (134 mmol NaCl/L, 2.9 mmol KCl/L, 0.34 mmol Na2HPO4/L and 2 mmol MgCl2/L) containing 0.2% of bovine serum albumin, 5 mmol glucose/L and 10 mmol HEPES/L, pH 7.35. The platelets that had been submitted to exclusion chromatography (PSEC) were adjusted to a final concentration of 2×1011 cells/L. Since the addition of methanol to the suspensions of PSEC at concentrations<0.5% did not cause any change in the response of the PSEC to collagen, this ratio was used for obtaining final concentrations of quercetin that varied between 5 and 20 μmol/L. Catechin and quercetin were added to the suspensions of PSEC while stirring continuously for 30 min at 37° C. and then removed by centrifugation at 800×g for 20 min at room temperature.

Quelle