*2.6. Proliferation and LDH Release*

Acute cell damage upon transfection was determined by measuring the lactate dehydrogenase (LDH) release, using the Cytotoxicity Detection Kit from Roche (Mannheim, Germany) according to the manufacturer's protocol. In brief, 35,000 cells were seeded in a 24 well plate. One hour prior to transfection, the medium was replaced with fresh medium and the cells were transfected with the polyplexes as described above containing 0.4 μg siRNA. After 24 h, the medium was harvested. Medium from untreated cells served as negative control and medium of cells treated with Triton X-100 (2% final concentration) was used as positive control (= 100% value) and fresh medium was included for the background value. In a 96 well plate, 50 μL medium was mixed with 50 μL reagent mix and incubated for 30 min in the dark at room temperature. The absorbance was measured at 490 nm and 620 nm as a reference filter in a plate reader (Thermo Fisher, Schwerte, Germany). Acute cytotoxicity values are presented as a percentage of the positive control after subtracting the background.

For the determination of the cell viability/metabolic activity 10,000 cells in 100 μL medium were seeded per well of a 96 well plate. The following day, the cells were transfected with polyplexes at different amounts as indicated in the figure. Numbers of viable/metabolically active cells were measured 72 h after transfection by using the colorimetric WST-8 Cell Counting Kit (Dojindo Molecular Technologies EU, Munich, Germany). Briefly, after replacing the medium with 50 μL of a 1:10 dilution of WST-8 in serum-free medium, the cells were incubated for 1 h at 37 ◦C. The absorbance was measured at 450 nm and 620 nm as a reference wavelength in a plate reader.
