Neutrophil Isolation from PICU Patients

Blood was collected from participants during their PICU admission through an existing peripheral intravenous catheter with blood return or a central venous catheter into an ethylenediaminetetraacetic acid (EDTA) vacutainer tube. Tubes were centrifuged at 400ΧG to separate cells from platelet-rich plasma. Pelleted blood cells were resuspended in sterile phosphate buffered saline (PBS; ThermoFisher Scientific, Waltham, MA) with 2.5 mM EDTA up to the original whole blood volume. Neutrophils were negatively selected from PBS-EDTA washed whole blood using the EasySep Direct Human Neutrophil Isolation kit (StemCell Technologies, Cambridge, MA) according to the manufacturer’s protocol. Neutrophil purity was > 99% as confirmed by microscopic analysis of a Diff-Quik stained CytoSpin sample of purified neutrophils. Two million neutrophils were resuspended in 1 mL of RNALater and stored at − 80 °C until batch RNA purification.