12 ml polypropylene tubes

Frozen (–80°C) EDTA blood samples (3 ml) were thawed on ice and incubated for 10 minutes with 5 volumes of red blood cell lysis buffer (15.5 mM NH₄Cl, 1 mM KHCO₃ and 0.01 mM tetra-sodium EDTA in di-ethyl-pyrocarbonate treated deionized water; pH 7.3). Cells were centrifuged (10 minutes at 600 g) and pellets were incubated with 1.5 ml fresh buffer on ice for another 10 minutes. Blood leukocytes were obtained by centrifugation (5 minutes at 600 g). Ejaculate leukocytes were isolated from frozen-stored ejaculate pellets by density gradient centrifugation. Briefly, ejaculate pellets were thawed on ice and dissolved in 600 μl DMEM-F12 medium (Gibco). Samples were then placed on top of a pre-layered Histopaque^®1077 gradient (1.5 ml 90% + 1.5 ml 50% + 1.5 ml 20%; all solutions at 4°C) in 12ml-polypropylene-tubes (Sarstedt) and centrifuged (30 minutes at 400 g). The phenol-red from DMEM-F12 allowed for convenient identification of resulting phases. Upper fraction contained leukocytes, and the pellet contained sperm cells. Leukocytes were pelletized by centrifugation for 5 minutes at 600 g and 4°C. Leukocytes from blood and ejaculates were washed twice with PBS and frozen at –80°C until DNA and RNA isolations.