Fluorochrome 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),... 

Because multiple photodetectors are available, a flow cytometer has the ability to measure two or more fluorescence signals simultaneously from the same cell. To use several fluorochromes at the same time, cytometrists with only one laser required a group of stains, all of which absorb 488 nm light but which have different Stokes shifts so that they emit fluorescent light at different wavelengths and thereby can be distinguished from each other by the color of their fluorescence. Propidium iodide and fluorescein are a pair of fluorochromes that fulfill these criteria (having different Stokes shifts) and can be... 

Fig. 8.24. Cells in late apoptosis have fragmented DNA. They can be visualized as cells with increased incorporation of fluorochrome-labeled nucleotides to the ends of the fragments in the flow cytometric TUNEL assay (lower dot plots, with FL1 as FITC-dUTP and FL2 as propidium iodide bound to DNA). Alternatively, they can be visualized as cells with less-than-normal (sub-GO/Gl) DNA content (upper histograms of propidium iodide fluorescence). Data from etoposide-treated ML-1 cells courtesy of Mary Kay Brown and Alan Eastman.

Annexin V Annexin V is a molecule that binds to phosphatidylserine and, therefore, if conjugated to a fluorochrome, will identify apoptotic cells (which express phosphatidylserine on their surface). In the assay for apoptosis, annexin V must be used in conjunction with propidium iodide in order to exclude dead cells (which express phosphatidylserine on the internal side of their cytoplasmic membranes). 

Fluorochrome A fluorochrome is a dye that absorbs light and then emits light of a different color (always of a longer wavelength). Fluorescein, propidium iodide, and phycoerythrin, for example, are three fluorochromes in common use in flow cytometry. Flu-orophore is an equivalent term. 

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). 

Fluorochromes can be attached to antibodies which will then bind to specific chemical structures on or inside cells. Many other chemical and physical properties of fluorochromes determine when and where these dyes are useful in various biological assays. For example, some of the fluorochromes that bind to DNA, such as Hoechst 33342, can get into living cells, but most DNA-binding fluorochromes cannot get past the cell membrane. Those fluorescent dyes that cannot get past an intact cell membrane, such as propidium iodide (PI), are often used to distinguish live from dead and dying cells. 

At the cellular level cisplatin causes a block in the cell cycle at the point where DNA repair occurs, the S/G2 interface. The stages of the cell cycle can be monitored using flow cytometry75. In this technique cells are fixed and stained with propidium iodide, a DNA fluorochrome, which binds quantitatively to DNA. The degree of fluorescence is therefore directly proportional to the DNA content of the cell, which in turn is an indicator of the position of the cells in the cell cycle. The ovarian cell line, SK-OV-3, was incubated with [Au(acetato)2(damp)] for 4 hours at concentrations of either 30 pM or 100 pM and cell cycle analysis determined from DNA content. Using this technique it was apparent that the gold(III) complex [Au(OAc)2(damp)], unlike cisplatin, did not exert a cell cycle specific effect69. 

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