Isolation and Stimulation of Murine Neutrophils

We used a previously published VavCre/Jak2^V617F murine model that results in constitutive heterozygous expression of the Jak2^V617F activating mutation (18 (link)). Peripheral blood was collected from 10 to 12-week-old mice via the retroorbital venous plexus and was processed within 2 hours. Red blood cells were first sedimented with Hetastarch (6% hydroxyethyl starch, HES) in 0.9% NaCl solution at 1:4 v/v dilution at 37^oC. Next, supernatant was collected and subjected to brief hypotonic lysis with sterile water. Neutrophils were isolated by negative selection with magnetic beads according to manufacturer’s instructions (Neutrophil Isolation Kit, mouse, Miltenyi Biotec) and resuspended in 2% heat-inactivated FBS. Neutrophils were plated at 10–15,000 cells per well in 96-well optical-bottom plates in triplicates (Nunc MicroWell 96-Well Optical-Bottom Plates, ThermoFisher Scientific). Cells were then stimulated with ionomycin 4 μM (Sigma-Aldrich) or PMA 10 and 100 nM (Sigma-Aldrich) for 2.5 hours. Cells were then instantly fixed in 2% PFA and stained as described below.