TranslationBlocker Human Caspase-8 siRNA – DISCONTINUED

DISCONTINUED: Human Caspase-8 TranslationBlocker™ siRNA Duplex Product Attributes

Caspase-8 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. For the above data 6 well plates were seeded with 1×10⁵ cells and incubated at 37°C overnight.
   2. Prepare transfection reagent in serum-free growth medium such as Opti-MEM-I. Allow to come to room temperature. Lipofectamine 2000 was used to generate the above data in accordance with manufacturer’s instructions.</ li>
   3. Dilute siRNA to working concentration in serum-free growth media. For Lipofectamine 2000 or similar transfections into 6 well plates, we recommend starting siRNA dose optimization at around 100-125 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 Lipofectamine 2000 or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 20-30 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells. For 6 well plates this will entail adding the solution to cells in approximately 2.5 ml serum-free medium. 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 in serum free media. Typically, 4 hour incubation is recommended for this initial transfection step.
   7. Add serum containing media to culture without a media change if longer incubation times are required. Generally 1-2 fold of the original volume of siRNA containing serum-free media is added to restore nutrient levels in media. Alternatively, the media may be directly supplemented with serum such as 10% FBS.
   8. Incubate 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. 48 hours post-transfection, cells were treated with TRAIL (Catalog Number QP5244, 100 ng/ml), IL-1beta (Catalog Number QP5290, 10ng/ml), and TNF alpha (Catalog Number QP1385, 10ng/ml). After 10 hours, cells were lysed and Caspase-8 knockdown at the protein level was measured via Western Blot.