Chromatin condensation detection

On the other hand, there is strong evidence that the effects of flavonoids on cultured cells are not merely toxic but via induction of apoptosis or inhibition of proliferation. Thus signs of apoptosis, such as induction of caspase-3, fragmentation of DNA and chromatin condensation, are frequently detected upon cell exposure to flavonoids [213, 217-221]. In addition, the effects of flavonoids are generally reversible upon removal or addition of serum [222-224]. 

Currently, there are many methods available for determining cell death by apoptosis in cell cultures and tissues. These methods are based essentially on changes that occur in apoptotic cells. During apoptosis, several phenomena can be observed, such as DNA fragmentation, chromatin condensation, nuclear fragmentation, cytoplasm acidification, cytochrome c release from the mitochondria, exposure of intracellular phospholipids and the activation and breakdown of proteins. These apoptotic phenomena can be detected by direct or indirect methods, on cell populations or on individual cells that are representative of a population. The main principles used by the different detection methods are ... 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

Several methods are based on microscopy observations to detect morphological changes in the cells, mainly chromatin condensation. Different markers can be used to differentiate between the stages of apoptosis. 

Fig. 2. Identification of apoptotic cells by LSC based on high values of maximal pixel detecting red fluorescence or fractional DNA content of propidium iodide (PI) stained cells. Exponentially growing human leukemic HL-60 cells, untreated (A) or induced to undergo apoptosis by treatment with camptothecin (B) (refs. 26,28), were stained with PI in the presence of RNase as described in the protocol. The scatterplots represent bivariate distributions of cells with respect to their integrated red fluorescence (proportional to DNA content) vs maximal red fluorescence pixel value. Only mitotic cells (M) have high maximal pixel value in the untreated culture. Apoptotic cells (Ap) that are present in the CPT treated cultures, are characterized either by the increased intensity of maximal pixel of red fluorescence or by a low tsub-Gj) DNA content. The relocation feature of LSC allows one to observe morphology of the cells selected from particular regions of the bivariate distributions. Upon the relocation, the cells with high maximal pixel value or with fractional DNA content show chromatin condensation and nuclear fragmentation, typical of apoptosis (panels on right).

Milgrom and co-workers [105] recently developed an immunogold method for detection of PR in the rabbit uterus and have examined the effect of hormone addition on receptor localization at the ultrastructural level. PR were found to be predominantly nuclear in the presence and absence of hormone, but a small amount was detectable in the cytoplasm which was not apparent at the light microscopical level. These cytoplasmic PR were localized over endoplasmic reticulum and clusters of free ribosomes and may likely represent newly synthesized protein. No PR were located in the plasma membrane. Within the nucleus, unoccupied PR were associated with condensed chromatin which became more dispersed after hormone addition. These ultrastructural studies indicate that steroid-free PR translocate from their site of synthesis in the cytoplasm to the nucleus in a hormone independent manner, and that addition of hormone changes their intranuclear localization. 

Fig. 5. Scattering patterns of CE nuclei, a Scattering pattern of nuclei-EDTA. Bands indicated by arrows are the 0.025 nm" interfibre interference, the 0.06 nm intemucleosomal interference and the 0.156, 0.27 and 0.36 nm" bands which are due to the contributions from the nucleosome core particles, b Scattering pattern of nuclei at high ionic strength (150 mM NaCl). The 0.05 nm band is no longer visible but a weak band at 0.045 nm is detected. Arrows indicate the interfibre interference and the intranucleosomal bands as well as the 0.083 nm" and 0.15 nm" bands which result from the close approach of nucleosomes in the condensed chromatin fibre (Fig. 3 a) fi om Bordas et al., 1986 a)...

Significantly, all three of these DNA-specific staining protocols have pictures of intranuclear DNA distribution very similar to that obtained by prior detergent premeabilization in appropriate isolation buffers (Belmont etal., 1989)—namely, the absence of DNA stain from the euchromatic compartment, and the packaging of most DNA within condensed structures well above the 30-nm chromatin fiber size. More impressively, both the osmium ammine and the chemical pretreatment (NAMA-Ur) methods have the sensitivity to detect mitochondrial and viral DNA... 

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