Sodium heparin blood collection tubes

Whole blood (~350 mL) was collected from both male and female healthy adult donors via antecubital venipuncture into BD vacutainer, and Sodium Heparin blood collection tubes (BD, Franklin Lakes, NJ). Blood was diluted 2-fold in calcium- and magnesium-free phosphate-buffered saline (PBS pH: 7.2; Fisher Scientific, ​​Waltham, MA) within 30 min of collection. A single-step density gradient centrifugation was performed to isolate peripheral blood mononuclear cells (PBMCs) by slowly layering diluted blood (30 mL) over 15 mL of Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) contained within either a 50 mL SepMate™ (STEMCELL Technologies) or a 50 mL conical tube. Stepwise centrifugation at 1,200 x g for 10 min at room temperature with slow deceleration was performed. The buffy coat interface containing PBMCs was collected into 50 mL conical tubes and pelleted via centrifugation (1,400 RPM for 10 min). Residual red blood cells were removed with treatment with ACK Lysing Buffer (Gibco, Waltham, MA). PBMCs were then washed twice with 1X PBS, pelleted as above, and cryopreserved at -80°C in 90% (v/v) fetal bovine serum (FBS; R&D Systems, Minneapolis, MN) and 10% (v/v) dimethyl sulfoxide (Corning, Corning, NY) in cryovials labeled with each donor’s unique sample identification number, followed by transfer to liquid nitrogen storage within 24 h.

Approximately 20 ml of blood was collected from each donor into sodium heparin blood collection tubes (BD; Franklin Lakes, NJ, USA). Peripheral blood mononuclear cells (PBMCs), from 12 healthy donors, were separated from heparinized blood using Ficoll-Hypaque density gradient [16 ] and were suspended in complete RPMI (RPMI-1640 medium supplemented with 10 mM HEPES, 2 mM L-glutamine, 10% Fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin).
The study was approved by the ethical review committees of faculty of medicine, Thammasat university under number 119/2555.