5810 r swing bucket plate rotor

PMNs (120 μl of 5 × 10⁶/ml) were added to 480 μl aEV, lysed aEV, sEV, apoEV sample or HBSS at 37°C in a linear shaker (0.18 g) for 45 min. aEV, lysed aEV, and sEV samples were prepared from 1.92 × 10⁷ cells, whereas apoEV samples were derived from 0.96 × 10⁷ cells. The pretreated PMN samples were placed in the wells of a 3 μm pore Corning, NY, USA transwell cell culture plate coated with 10% FBS. Every well contained 2 × 10⁵ cells. As a chemoattractant, 100 nM N-formylmethionyl-leucyl-phenylalanine was used. After 1 h incubation at 37°C, the transwell plate was centrifuged (Eppendorf 5810 R swing-bucket plate rotor, 3,220 g, 3 min, 4°C). Transmigrated cells were counted using an acid phosphatase assay^{37 (link)} in a plate reader (Labsystems iEMS Reader MF; Thermo Scientific).