DNA staining

In Pins - embryos the initiation steps of apical complex formation occur normally. However, this complex cannot be maintained in mitotic neuroblasts. Hence, the importance of the maintenance of this complex for asymmetric cell division can be ascertained by assessing how Pins- neural progenitors divide. Pins- embryos exhibit all of the defects seen in insc mutants. Mitotic spindle orientation is defective. In the cells of mitotic domain 9 the 90° reorientation, which normally occurs in wild-type resulting in the orientation of the spindle along the apical—basal axis (Fig. 3A), fails to occur in the mutant (Fig. 3B). Mitotic spindle orientation of neuroblasts in the segmented CNS, deduced from DNA staining, also often fails to... 

Thomas, L, Netzel, K. N. and Zhao, M. (1995). Base-content dependence of emission enhancements, quantum yields, and lifetimes for cyanine dyes bound to double-strand DNA Photophysical properties of monomeric and bichromophoric DNA stains. J. Phys. Chem. 99,17936-17947. 

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

This protocol uses propidium iodide (PI) as the fluorescent tracer for DNA content (3-6). PI binds to both double-stranded DNA and double-stranded RNA. Therefore, RNase will be used to reduce the double-stranded RNA resulting in only DNA staining. For alternative DNA-specific ligands and dyes, see Chapter 30. 

Finally, flow cytometric DNA staining has been regarded as an objective prognostic parameter in several types of human cancer, but it is important to remember that this is a snapshot of the cell cycle. From a DNA-content histogram, it cannot be determined if a cell is actively moving through the cell cycle, has slowed, or even stopped its traverse through the cell cycle. 

Leakage of cell contents (e.g., lactate dehydrogenase) and entry of extracellular dyes (e.g., Trypan blue, DNA stains such as TOTO-3)... 

Cell number frequency distribution of nuclear DNA content of cell population, protein content, protein synthesis (e.g., 14C-labeled methionine incorporation), DNA synthesis (e.g., tritiated thymidine incorporation), DNA stains (e.g., Hoechst 33 342 4, 6-diamidino-2-phe-nylindole, DAPI picogreen) mass tracker dyes (e.g., LysoTracker green for lysosomes, MitoTracker deep red for mitochondria, ER-Tracker blue-white DPX for endoplasmic reticulum)... 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

FIG. 1. Expression of clock proteins in mouse SCN. (a) Immunostaining of mouse SCN sampled at beginning (CTO) and end (CT12) of circadian day reveals constitutive expression of mCLOCK, and rhythmic expression of mCRY (scale bar 500 /tm). Inset high power confocal views of mCLOCK-ir and Hoescht DNA stain confirm nuclear localization of mCLOCK-ir. 

A fundamental prerequisite for detection of intranuclear antigens by FCM is fixation and/or permeabilization to facilitate access of antibodies to their epitopes. Since most studies will be earned out in conjunction with DNA staining, the preparation and staining procedures must meet the following criteria ... 

Fig. 2 Left cytoplasmic microtubules in interphase kidney epithelial cells imaged with the fluorescent paclitaxel derivative Flutax-2 (green) and nuclear DNA stained with Hoescht 33342 (blue). Right mitotic spindle from a dividing metaphase cell with similarly imaged microtubules and chromosomes. Bars indicate 10 pm (micrographs courtesy of Isabel Barasoain)...

Examine under the fluorescence microscope when DNA stains greenish/yellow. 

If one chooses PI for DNA staining, the following procedure may be used.7 After the nuclei are pelleted, they are resuspended in staining solution, which consists of 3% (w/v) polyethylene glycol (PEG) 6000, 50 pig/ml PI, 180 units/ml RNase, 0.1% Triton X-100 in 4 mM citrate buffer. The pH of the staining solution is 7.2. The nuclei are then incubated at 37° for 20 min. After incubation, an equal volume of salt solution is added. The... 

Gamer, D.L. et al. 1994. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and pro-pidium iodide. J. Androl. 15, 620-629. 

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