Ficoll paque plus

Following collection into a BD EDTA Vacutainer, 5 ml of blood was pipetted and diluted with 5 ml of sterile phosphate buffer saline (PBS) and underlayed with 5 ml of Ficoll Paque PLUS in a 15-ml Falcon tube. It was centrifuged at 1,800g at room temperature for 20 min. Peripheral blood mononuclear cells were aspirated into a 15-ml Falcon tube before addition of 6 ml of 1 × BD Pharm Lyse to remove red blood cells. After incubation for 15 min at room temperature the lysis solution was diluted to 15 ml with RPMI+10% fetal bovine serum (FBS) and tubes centrifuged at 300g, at 4 °C for 5 min. The cell pellet was resuspended into 10 ml of RPMI+10% FBS and centrifuged at 300g, at 4°C for 5 min. Cells were finally resuspended in 500 μl of RPMI+10% FBS and a 10 μl sample taken for counting using a MACSQuant. Cells were then adjusted to the appropriate concentration for use in experiments as per the relevant methods section. Cell concentration was adjusted to 5 × 10⁶ cells per ml with RPMI+10% FBS. The samples were aliquoted into sterile 96-well U-bottom plates and the remaining cell suspensions were centrifuged at 300g, 4 °C, 5 min. The supernatant was aspirated up to ∼100 μl mark and 300 μl of Trizol LS reagent was added before storage at −80 °C. This preparation of neutrophil samples possibly has a contamination of eosinophils of ∼1–4% and 1–2% of lymphocytes.