Cell dyn 3500 system

Venous blood (20 mL) was collected
from healthy human subjects and
gently mixed with 1 mL of anticoagulant (0.15 M EDTA). Platelet-rich
plasma was obtained by low-speed centrifugation (200g for 20 min at 22 °C). Platelets were counted automatically
with a flux cytometer (Cell-dyn 3500 system; Abbott, Milano, Italy).
Written consent was obtained from all subjects, and the study was
approved by the local Ethics Committee.
For measurement of the
[³H]5-HT uptake, platelets were used immediately; whereas
for [³H]paroxetine binding, platelets were precipitated
by centrifugation at 10,000g for 10 min at 4 °C,
and the pellets were then stored at −80 °C until the assay.
For human platelet membrane preparation, platelet pellets were
washed with 10 mL of 50 mM Tris-HCl buffer, pH 7.4, containing 150
mM NaCl and 20 mM EDTA. Pellets were lysed and homogenized in 10 mL
of 5 mM Tris-HCl buffer, pH 7.4, containing 5 mM EDTA and protease
inhibitors (200 μg/mL bacitracin, 160 μg/mL benzamidine,
and 20 μg/mL soybean trypsin inhibitor) using an Ultra-Turrax
homogenizer and centrifuged at 48,000g for 15 min
at 4 °C. The resulting pellets were resuspended in 50 mM Tris-HCl
buffer, pH 7.4, containing 120 mM, NaCl, and 5 mM KCl (assay buffer).
Protein concentration was determined according to the method of Lowry
et al.^{59 (link)} after solubilization in 0.75 M
NaOH and using bovine serum albumin (BSA) as standard.

At the end of the study, the rats were euthanized by overdose inhalation of carbon dioxide. Blood samples were collected by cardiac puncture and rats were humanly killed by exsanguinations. Hematological and blood clinical chemistry tests were conducted by an ABBOTT CELL-DYN 3500 system (ABBOTT Laboratories, IL, USA) and a Hitachi 902 automated blood analyzer (Hitachi Science Systems Ltd., Ibaraki, Japan).