3.5. DNA Fragmentation Assay

Fragmentation of genomic DNA was determined by means of the Cell Death Detection ELISAPLUS kit (Roche, Basel, Switzerland). This method rests on the immunoenzymatic detection of DNA–histone complexes in the cytoplasmic cell fraction or cell culture supernatants. Cells were plated and cultured in 96-well plates (Greiner Bio-One) at the initial density of 5 × 10³ cells per well in 100 µL of culture medium. Cells were allowed to attach and grow for 1 day prior to the addition of test reagents. Then, the media were replaced with the working solutions (3 µM, 10 µM, 30 µM), and cells were exposed to compounds 2–6 for 24 h. In some cases, 5 µM cyclosporin A (CsA, an inhibitor of the mitochondrial permeability transition) was added to the cells 30 min before treatment with the betulin derivative. Subsequently, cells were incubated concomitantly with both compounds. After a 24 h incubation period, plates were centrifuged (200× g; 10 min), media were removed, and cells were lysed for 30 min at room temperature. At the next step, the lysates were again spun (200× g; 10 min), and 20 µL of supernatants (cytoplasmic fractions) was moved to the new 96-well streptavidin-coated plate. In one experiment, the presence of mono- and oligonucleosomes was determined in 20 µL samples of centrifuged culture supernatants (media) to detect the permeability of plasma membrane (considered as an indicator of cell necrosis). Then, the sandwich ELISA assay was fulfilled according to the manufacturer’s protocol. Absorbance was measured at 405 nm and 490 nm (reference wavelength), and the enrichment factor of the cytoplasmic (or supernatant) fraction in mono- and oligonucleosomes was calculated according to the formula:

A high EF index in the cytoplasmic fraction (resulting from DNA fragmentation while maintaining the tightness of cell membranes) is a typical indicator of the relatively early stage of apoptosis.