Blood Collection and Isolation of Cells:
Peripheral venous blood samples were collected from each participant by
venipuncture into heparinized syringes and processed immediately. To
isolate peripheral blood monocytes (PBMCs), blood was diluted 1:1 with
phosphate-buffered saline (PBS, 10 mM
NaH2PO4, 2 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl; pH 7.2)
and 30 mL were layered over 15 mL of Ficoll-Paque density gradient and
centrifuged at 500 g for 20 min. The interface layer (buffy coat) was
then collected. Cells were washed twice with PBS and adjusted to
1×106 cell/mL in HEPES
(4-(2-hydroxyethyl)-1-piperazine) ethanesulfonic acid buffered saline
containing 15 mM HEPES, 125 mM NaCl, 6 mM KCl, 1.2 mM
MgSO4, 1.0 mM NaHCO3, 1.0 mM
CaCl2, 10 mM glucose; pH 7.4).