Proliferation and apoptotic cell death assessment
Proliferation, apoptosis and RNA expression were investigated 48h post-
transfection. Proliferation was assessed by colorimetric detection based
on 5-bromo-2’-deoxyuridine (BrdU) incorporation (Roche, Mannheim,
Germany), according to manufacturer’s protocol. Briefly, cells were
incubated with BrdU for 3h, and absorbance was read at 370nm (SpectraMax
Plus 384 Microplate Reader, Molecular Devices LLC, San Jose, CA, USA)
48h post- transfection. Cells incubated for few seconds with BrdU- were
used as background control.
Apoptosis was measured 48h post- transfection by photometric detection
of cytoplasmic histone-associated-DNA-fragments in cell lysates
according to manufacturer’s instructions (Roche). Briefly, cell lysates
were loaded on to a streptavidin-coated plate and subsequently incubated
with biotinylated anti-histone antibodies and anti-DNA antibodies
conjugated with peroxidase (POD). Unbound antibodies were removed by
washing. Substrate solution, tetramethyl-benzidine (TMB) (Roche), was
added. The peroxidase activity in the immunocomplex was determined
photometrically at 405nm.