**2.8. DNA fragmentation analysis**

Qualitative DNA fragmentation (Roche) kit was used in this analysis. Briefly, after trypsinization, 1 × 10<sup>6</sup> cells were collected and washed twice with PBS. The cells were then pelleted by centrifugation at 3000 rpm for 5 min, the supernatant removed and replaced with 200 μL of fresh PBS. The sample was thereafter lysed with 200 μL binding/lysis buffer consisting of 6 M guanidine-HCl, 10 mM urea, 10 mM Tris–HCl, 20% Triton X-100 (v/v), pH 4.4 and incubated for 10 min at 15–25°C. After the incubation, 100 μL of isopropanol was added and the mixture was filtered through a high pure spin filter tube combined with a clean collection tube and centrifuged at 8000 rpm for 1 min. The flow-through was discarded and the filter tube was combined again with the collection tube before washing with 500 μL of washing buffer consisting of 20 mM NaCl, 2 mM Tris–HCl, pH 7.5 in 20 mL with the addition of 80 mL ethanol. This washing step was done twice before spinning the tube dry at 13,000 rpm (Eppendorf 5424 microcentrifuge, USA) for 10 s to remove any residual washing buffer.

The extracted DNA was eluted into a fresh collection tube with 200 μL of prewarmed (70°C) elution buffer (10 mM Tris) and recentrifuged at 8000 rpm for 1 min. The quality of the extracted DNA was assessed with a nanodrop spectrophotometer (nanodrop lite spectrophotometer, Thermo Scientific, USA). The DNA sample was mixed with 1× loading dye and electrophoresed on 1% agarose gel 75 V for 1 h and stained with GelRed™ nucleic acid gel stain. The fragmented DNA was visualized under UV transilluminator and photographed with a chemiluminescence image analyzer system (Chemi-Smart, Vilber Lourmat, Germany).
