*2.4. Luciferase Assay and Flow Cytometry*

Knockdown efficiencies were analyzed by measuring reporter gene activities (luciferase or enhanced green fluorescent protein, EGFP) 72 h after transfection. For the determination of luciferase activity, the medium was aspirated and the cells were lysed with 300 μL Luciferase Cell Culture Lysis Reagent (Promega, Mannheim, Germany) for 30 min at room temperature. In a test tube 10 μL cell lysate was mixed with 25 μL luciferin reagent (Beetle-Juice Kit, PJK, Kleinblittersdorf, Germany) and immediately measured in a luminometer (Berthold, Bad Wildbad, Germany).

EGFP expression levels were determined by flow cytometry. The cells were harvested by trypsinization and centrifuged for 3 min at 3,000 rpm, prior to resuspension of the cell pellets in FACS buffer (0.5 mL PBS, 1% FCS, 0.1% NaN3). 20,000 cells in the vital gate were measured in an Attune® Acoustic Focusing Cytometer (Applied Biosystems, Foster City, CA, USA).
