Roswell park memorial institute 1640 medium

Human neutrophils were isolated from the peripheral blood of healthy volunteers, who provided written informed consent, using Lympholyte-poly (CEDARLANE Laboratories) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell lysis buffer (BioLegend), then washed twice in Hank’s buffered salt solution without Ca²⁺/Mg²⁺ (Wako Pure Chemicals), and finally resuspended in the Roswell Park Memorial Institute 1640 medium (Wako).
Neutrophils from micro mini pigs were isolated using previously described methods,²⁴ (link) with modifications. Blood from the pulmonary artery of micro mini pigs was collected in a container coated with EDTA-2K and treated with red blood cell lysis buffer for 10 minutes at 4 °C. Neutrophils were separated from mononuclear cells by layering 5 mL of the cell suspension on 5 mL of Percoll 1.081 (GE Healthcare), which was layered under 5 mL of Percoll 1.087, followed by centrifugation at 1,000g for 20 minutes at 25 °C. The middle layer, enriched with neutrophils, was collected and washed twice in Hank’s buffered salt solution without Ca²⁺/Mg²⁺. The cells were then resuspended in red blood cell lysis buffer and washed twice with Hank’s buffered salt solution without Ca^2+/Mg²⁺.