Isolation of Monocytes and Neutrophils

Peripheral blood was collected in two 10 ml sodium heparin tubes (14–18 ml of blood). Red blood cells were depleted with 1 volume HetaSep (Stem Cell Technologies) for 5 volumes whole blood and centrifuged at 92g, 6 min, no brake. The nucleated cells were layered on Histopaque 1077 (Sigma) and centrifuged (400g, 30 min, no brake). PBMCs were washed once with PBS before isolating cell types using magnetic microbeads. CD14+ monocytes were isolated with the Human CD14 Positive Selection Kit as previously described (EasySep™, Stem Cell Technologies) [50 (link), 51 (link)]. RNA was extracted from monocytes with the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Chatsworth, CA). In parallel, neutrophils were isolated from the red blood cell/granulocyte pellet remaining after the Histopaque spin. The neutrophil pellet was washed once with PBS, and red cells were lysed with 5 ml water followed by 5 ml 2× PBS. Neutrophils were pelleted (300g, 10 min), lysed with STAT-60 (Tel-Test “B”, Friendswood, TX, USA), and RNA was extracted.