TranslationBlocker Human Interleukin-10 / IL-10 siRNA

Human Interleukin-10 / IL-10 TranslationBlocker™ siRNA Duplex Product Attributes

Interleukin-10 / IL-10 Knockdown Protocol:
1. 1. Allow PBMC to adhere for 2h at 37°C. Wash away non-adherent cells and generate DC by culturing in the presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml) for 6 days. On day 6, begin transfection protocol
   2. Prepare transfection reagent in serum-free growth medium such as Opti-MEM-I. Allow to come to room temperature. GeneSilencer Transfection reagent was used to generate the above data in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For GeneSilencer or similar transfections into 12 well plates, we recommend starting siRNA dose optimization at around 100-150 pmol siRNA duplex.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Ratio of transfection reagent to siRNA should be optimized. Equivalent volumes of prepared GeneSilencer or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 30 minutes with gentle mixing (optional). GeneSilencer was selected or this experiment to avoid serum starving primary DC culture as it is compatible with serum containing media.
   5. Add prepared siRNA solution to cells. For 12 well plates this will entail adding the solution to cells in approximately 1.1 ml growth media. Optionally, FITC labeled control siRNA (Product Number QC-1F) or positive control siRNA (Beta-Actin; Product Number QC-2) may be used to assess transfection efficiency.
   6. Incubate for several hours before a media change. The above data was generated with an initial 24 hour incubation.
   7. Incubate further and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. IL10 knockdown at the protein level was measured at 48 hours post transfection via Western Blot.