DNA fragmentation detection using DNA laddering assay

A DNA ladder assay was performed to identify DNA fragmentation among apoptotic cells as previously described [12]. Briefly, 1 × 10⁶ cells/well were seeded into a 24-well, flat-bottomed plate and incubated in a 5 % CO₂ incubator at 37 °C for 24 h. After treatment of cancer cells with 2 × IC₅₀ of the 50 % ethanol–water extracts or melphalan for 24 h, the cells were collected and washed with medium. The DNA in the cell pellets was extracted using a FlexiGene DNA kit. Aliquots (2 μg) of the DNA were analyzed using electrophoresis at 100 V for 40 min in 1.8 % agarose gels containing 0.1 % ethidium bromide. After electrophoresis, the DNA fragments were visualized using a UV transilluminator (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) and gel pictures were taken with a UV-illuminated camera (Syngene, UK).