Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells [post]

Pavlina Majtnerova, Jan Capek, Filip Petira, Jiri Handl, Tomáš Roušar
2021 unpublished
At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods has used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in intact cells. We used HepG2 and HK‑2 cells cultured in 96-well plates which were treated with potent apoptotic
more » ... h potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Then, 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 apoptosis (i.e. TUNEL, DNA ladder, caspase activity). We found that the developed assay provides results of same sensitivity as TUNEL assay but the advantages of the spectrofluorometric assay are fast processing, low-cost and 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.
doi:10.21203/rs.3.rs-208670/v1 fatcat:3nue4gj4xjcqvn2djd3kmowymy