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
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- bisbenzimidazol chemie MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- cisplatina farmakologie MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- fragmentace DNA účinky léků MeSH
- kamptothecin farmakologie MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.
- MeSH
- 4-chlor-7-nitrobenzofurazan chemická syntéza metabolismus MeSH
- atopická dermatitida metabolismus MeSH
- bisbenzimidazol metabolismus MeSH
- ceramidy chemická syntéza metabolismus MeSH
- fluorescence MeSH
- fluorescenční barviva chemická syntéza metabolismus MeSH
- kůže metabolismus MeSH
- lidé MeSH
- permeabilita MeSH
- psoriáza metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH