siRNA-Mediated Silencing of HCAR3 in Monocytes

Freshly isolated human monocytes were transfected with siRNA specifically targeting HCAR3 (OriGene), as shown in Stäubert et al. (2015) [50 (link)], using Viromer Green (Lipocalyx) according to the manufacturer’s protocol. 10 μL of siRNA-Viromer mix were preplated into a 96-well plate before the addition of freshly isolated monocytes resuspended in RPMI supplemented with 10% FBS (1.5 x 10⁵ monocytes/well) which were then incubated 14 h at 37 °C in a humidified 5% CO₂ incubator. cAMP assays were performed as described below. To monitor the transfection efficiency a red fluorescent siRNA (OriGene) and Hoechst 33342 (Sigma-Aldrich) were used. The viability of transfected monocytes was determined by staining with propidium iodide (PI) (Thermo Fisher Scientific) and Hoechst 33342. Transfection efficiency and viability were analyzed using the Celigo S Imaging Cytometer (Nexcelom). The transfection efficiency was found to be ≥ 80% with less than 50% of monocytes being PI-positive (S6 Fig).