Fluorescent labeling propidium iodide

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. 

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

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. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Membrane destabilization can be simultaneously monitored by using propidium iodide. This non fluorescent and poorly permeant small molecule enters into the cells only upon membrane permeabilization/ destabilization and becomes fluorescent upon binding to DNA in the nucleus. In the presence of melittin, cells are also rapidly (200 s) red-labeled upon peptide induced membrane permeabilization (Figure 16.4D). It can be seen that the diffusion of propidium iodide inside the cells is correlated with the leak of EGFP. The results show that melittin induces the formation of pores in the plasma membranes allowing the passage of... 

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.

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). 

The samples were processed and stained with propidium iodide according to the method described in Ref. [14.8]. Flow cytometry DNA analysis was performed using a FACScan cytometer. The fluorescence intensity of the DNA labelled with propidium iodide was processed using CellQuest software, and the histograms of the DNA content were analysed using ModFit software. For each sample, 20 000 events were acquired. The DI was calculated with reference to the DNA diploid from the same sample. 

Fig. 25. Bacteria (Bradyrhizobium japonicum) labeled with propidium iodide to define the bacterial volume (red) and a fluorescent antibody against BJ38 (FITQ green). Twenty-two optical sections (18 x 18)Um each) were imaged at 0.3/im intervals in the Z-axis and reconstructed with the SFP algorithm. Notice the polar organization of BJ38, a protein that is thought to play a role in attachment to soybean root hairs.

Fig. 7.6 Apoptosis observed by fluorescence microscopy. Apoptotic cells appear labeled with green or red fluorescence if very late apoptosis was achieved (Annexin V and propidium iodide staining). Necrotic cells appear labeled with red fluorescence. Left column images obtained with Nomarsky right column images obtained with fluorescence staining. Almost no apoptosis is de-...

Fig. 4. Cellular content of poly(ADP-ribose) polymerase by two-color fluorescent measurement. Human CEM T lymphoblastoid cells were fixed with 45% ethanol for 30 min and stained with (A) FTTC-labelled goat anti-human IgG, (B) FITC-labelled anti-poly(ADP-ribose) polymerase IgG, (C) normal human IgG followed by FTTC-labelled goat anti-human IgG or (D) affinity purified anti-poly(ADP-ribose) polymerase IgG followed by FTTC-labelled goat anti-human IgG (10 pg/ml in each experiment). Finally, cells were stained with propidium iodide (10 x g/ml) and two-color fluorescence was analyzed in a flow cytometer (11). Two-color contour plots shows DNA content (red fluorescence) on the X-axis, and HTC-antibody binding (green fluorescence) on the Y-axis. 

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