siRNA knockdown in RAW 264.7 cells

RAW 264.7 cells were passaged in RPMI Medium 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA USA #11875135) supplemented with 10% FBS at 37°C with 5% CO₂. 2.5×10⁵ cells were deposited onto glass coverslips in 6-well tissue-culture treated plates, centrifuged at 300×g for 5 min, and allowed to adhere via incubation at 37°C with 5% CO₂ for 1 h. Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA US #13387) was diluted in Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific #31985062), and mixed 1:1 with anti-Dectin-1, anti-CR3, or scramble Mouse Silencer Select siRNA (Thermo Fisher Scientific #430817) diluted in Opti-MEM Reduced Serum Medium per manufacturer recommendations. siRNA-lipid complexes were added to wells with RAW 264.7 cells at 12.5 pmol and incubated for 24 h at 37°C with 5% CO₂. Cells were then activated with 1 μg/mL IFN-γ, incubated for a further 24 h, and macrophage uptake assays were performed as above. To verify siRNA knockdown efficiency, CR3 or scramble siRNA knockdown was performed as described above, total RNA extracted, converted to cDNA, and finally measured via ΔΔCt RT-qPCR and expressed as a percentage of knockdown efficiency compared to the housekeeping gene GAPDH.