Isolation of Primary Human Lymphocytes

Buffy coats from healthy blood donors were received from the blood center at the Uppsala University Hospital, Sweden. Peripheral blood mononuclear cells (PBMC) were isolated using SepMate tubes-50 (Stem Cell Technologies) by density gradient centrifugation. Briefly, 10 ml Lymphoprep reagent (Stem Cell Technologies) was added to the tubes followed by addition of blood on top of Lymphoprep. The tubes were then centrifuged at 1200×g for 10 min. Next, cell suspension above the Lymphoprep was collected and PBMCs were washed twice with phosphate-buffered saline (PBS, Thermo Fisher Scientific). For optimal lysis of red blood cells, 5 ml ACK lysis buffer (Thermo Fisher Scientific) was added to the cells and incubated in the dark for 10 min at room temperature followed by centrifugation at 500 × g for 5 min. After that, primary monocytes were removed by an EasySep CD14+ selection kit II (Stem Cell Technology) according to the manufacturer’s instructions. Primary human lymphocytes were stored in −150 °C until use.