Isolating White Blood Cells from Whole Blood

To generate white blood cells (WBC), red blood cells were lysed from whole human blood, which was collected using either ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose (ACD) as a coagulant. Blood was mixed at a 1:10 ratio with ammonium-chloride‑potassium (ACK) lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4) and incubated for 5 min at room temperature. The WBCs were pelleted by centrifugation (500 ×g, 5 min) at room temperature and then washed with cold phosphate-buffered saline (PBS). For the isolation of human neutrophils, whole blood was mixed with hetasep (Stemcell) in a 1:5 ratio and incubated for 30 min to separate leukocytes from erythrocytes. Subsequently, the straw-colored layer of leukocytes was transferred to a fresh tube and neutrophils were isolated using the direct human neutrophil isolation kit (Stemcell) according to manufacturer's instructions. Both WBCs and purified neutrophils were finally diluted in complete R10 media (RPMI-1640 media (Sigma) with 10% FBS, 2 mM L-Gluthamine and 100 U/ml penicillin/streptomycin) for each assay.