At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

• MeSH
• Apoptosis drug effects   MeSH
• Bisbenzimidazole chemistry   MeSH
• Cell Death drug effects   MeSH
• Cell Nucleus drug effects   metabolism   MeSH
• Cell Line MeSH
• Hep G2 Cells MeSH
• Cisplatin pharmacology   MeSH
• Microscopy, Fluorescence methods   MeSH
• Spectrometry, Fluorescence methods   MeSH
• DNA Fragmentation drug effects   MeSH
• Camptothecin pharmacology   MeSH
• Humans MeSH
• Antineoplastic Agents pharmacology   MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Staurosporine pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bisbenzimidazole MeSH
• Cisplatin MeSH
• Camptothecin MeSH
• Antineoplastic Agents MeSH
• Staurosporine MeSH