Isolation and Enumeration of Neutrophils

Blood samples were collected in vacuum tubes containing an anticoagulant (0.004% EDTA) from the antecubital vein of the runners at rest. Neutrophils were obtained from whole blood diluted in phosphate-buffered saline (PBS, 1:1; pH 7.4 containing 100 mmol/L CaCl₂, 50 mmol/L MgCl₂) and carefully layered on histopaque (d = 1.077) gradient (43 (link), 44 (link)). The tubes were then centrifuged at 400 x g and 4°C for 30 min. The supernatant, rich in mononuclear cells, was separated. Neutrophils were prepared from the inferior sediment, and 10 mL lyse solution, containing 150 mmol/L NH₄Cl, 10 mmol/L NaHCO₃, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4, were added to promote lysis of contaminating erythrocytes. The preparation was homogenized and maintained in ice for 10 min to allow erythrocyte lysis. Afterward, the tubes were centrifuged at 400 x g and 4°C for 10 min, and this procedure was repeated to reduce residual erythrocyte. Cells were washed once with PBS. Neutrophils were then counted in a Neubauer chamber in an optical microscope (Carl Zeiss, Jena, Germany), according to the protocol used in our previous study (30 (link), 45 (link)).