In situ direct dna fragmentation assay kit

Apoptosis was analyzed by Annexin V-FITC Apoptosis Kit (Biovision). In brief, Claspin (f/+) and Claspin (f/-) MEFs were infected with Ad-Cre or untreated for 48 h. Cells were passaged and cultured for a further 24 h. Cells were then placed in serum-free medium and were harvested at 0, 24, and 48 h. Harvested cells were labeled with propidium iodide/RNase A solution and FITC conjugated annexin V and incubated in the dark for 5 min at room temperature. The labeled cells were analyzed by FACS. Apoptosis was analyzed also by TUNEL assay, in which DNA fragmentation was analyzed by using In Situ Direct DNA Fragmentation Assay Kit (Abcam) according to the manufacturer’s protocol. Briefly, 1 × 10⁵ Claspin(f/-) MEFs were infected with Ad-Cre or untreated for 48 h. Cells were passaged and cultured for further 24 h. Cells were then placed in serum-free medium for 48 h and harvested. Harvested cells were fixed with 1% paraformaldehyde and washed with PBS, followed by addition of 70% ice-cold ethanol for 30 min. After washing with wash buffer, cells were mixed with staining solution for 1 h at 37 °C and rinsed with the rinse buffer. After removing the rinse buffer by centrifugation, cells were re-suspended in propidium iodide/Rnase A solution and incubated in the dark for 30 min at room temperature. The FITC labeled cells were analyzed by FACS.