Alox15 trilencer 27 human sirna

Cells (1.5 3 10⁶) were subjected to electroporation using Neonâ Transfection System 100 mL Kit (Thermo Fisher Scientific). To maximize the efficiency, the Neon 24 optimization protocol was applied according to the manufacturer’s instruction which varied in pulse voltage, pulse width and the number of pulses. Macrophages, differentiated with M-CSF for 6 days, were harvested using PBS plus 1 mM EDTA and the cell pellet was resuspended in 1 mL of PBS. 20 mM of human non-targeting siRNA (Thermo Fisher Scientific; D-001810-01-05) and 20 mM of ALOX15 Trilencer-27 Human siRNA (OriGene, SR300171) was resuspended in the Resuspension Buffer T (Thermo Fisher Scientific) and added to the cells before electroporation. The electroporated cells were transferred immediately to a 12-well containing 1 mL of the corresponding growth medium plus 20 ng/ml IL-4 (Peprotech) and then incubated for another 48 h in a 5% CO₂ incubator.