Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells.

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.

Thin TiO2 Coatings by ALD Enhance the Cell Growth on TiO2 Nanotubular and Flat Substrates.

The present work exploits Ti sheets and TiO2 nanotube (TNT) layers and their surface modifications for the proliferation of different cells. Ti sheets with a native oxide layer, Ti sheets with a crystalline thermal oxide layer, and two kinds of TNT layers (prepared via electrochemical anodization) with a defined inner diameter of 12 and 15 nm were used as substrates. A part of the Ti sheets and the TNT layers was additionally coated by thin TiO2 coatings using atomic layer deposition (ALD). An increase in cell growth of WI-38 fibroblasts (>50%), MG-63 osteoblasts (>30%), and SH-SY5Y neuroblasts (>30%) was observed for all materials coated by five cycles ALD compared to their uncoated counterparts. The additional ALD TiO2 coatings changed the surface composition of all materials but preserved their original structure and protected them from unwanted crystallization and shape changes. The presented approach of mild surface modification by ALD has a significant effect on the materials' biocompatibility and is promising toward application in implant materials.

An overview of apoptosis assays detecting DNA fragmentation.

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.