Publication protocol
Transfection: The efficacy of eleven transfection reagents to promote uptake of the FITC-labelled siRNA was investigated. The bMDM were seeded at 1 × 105 cells/well in 24 well plates. After 3 h incubation at 37 °C, during which time the bMDM had adhered to the plastic, the cells were transfected with 50 nM siRNA. The transfection reagents; HiPerFect (Qiagen), INTERFERin (Polyplus), Lipofectamine RNAiMAX (Invitrogen), Lipofectamine 2000 (Invitrogen), DharmaFECT 1, DharmaFECT 2, DharmaFECT 3 and DharmaFECT 4 (Dharmacon), siPORT Amine (Ambion), X-tremeGENE (Roche) and N-TER (Sigma–Aldrich) were used according to the manufacturers’ instructions. After 16 h incubation at 37 °C the bMDM were harvested and analyzed by flow cytometry.
To investigate the efficacy of each siRNA to knock-down expression of the target genes, the bMDM were seeded at 2 × 105 cells/well in 24 well plates and, after 24 h incubation at 37 °C, were transfected with transfection reagent alone or with 50 nM siRNA. The transfection reagents were used at the optimum conditions determined by the previous experiments. To investigate any response of bMDM to the transfection reagents the bMDM were either incubated at 37 °C for 48 h or the medium was changed after 24 h and the bMDM cultured for a further 24 h before being harvested and the RNA extracted. To investigate MEFV knockdown, bMDM were incubated at 37 °C for 24 h, then the medium was replaced and bMDM were cultured for an additional 24 h. To stimulate MEFV expression bMDM were activated with 100 ng/ml Escherichia coli-derived lipopolysaccharide (LPS) (Sigma) and bMDM were harvested and RNA extracted 2 h post activation.
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