TranslationBlocker Human PD-L1 / CD274 siRNA

Human PD-L1 / CD274 TranslationBlocker™ siRNA Duplex Product Attributes

PD-L1 / CD274 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed 12 well plates with 5 x 104 cells and incubate at 37°C until cells reach 40% confluence
   2. Prepare transfection reagent in serum-free growth media such as Opti-MEM allow to come to room temperature. The above data was generated with a custom liposomal nanoparticle delivery system. Pilot experiments suggest RNAiMAX is also suitable for delivery of PD-L1 siRNA prepared in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For RNAiMAX or similar transfections, we recommend starting optimization at around 100 pmol siRNA duplex in 250 ul of serum free media. Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared RNAiMAX or similar and diluted siRNA should be combined and incubated at room temperature for 20 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells.
   6. Incubate for several hours in serum free media.
   7. Add serum containing media to culture without a media change. Generally 1-2 fold of the original volume of siRNA containing serum free media is added to restore nutrient levels in media.
   8. Incubate and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, as with the above data, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. PD-L1 knockdown at the protein level was measured at 48 hours post second transfection via flow cytometry.