Propidium

Propidium iodide (3,8-diamino-5-(3-diethylaminopropyl)-6-phenylphenantridinium iodide methiodide) [25535-16-4] M 668.4, m 210-230 (dec), pKeskd 4 (aniline NH2), pKesi(2) (EtN2). Recrystd as red crystals from H2O containing a little KI. It fluoresces strongly with nucleic acids. [Eatkins J Chem Soc 3059 7952.] TOXIC. 

One of the early events of the apoptotic process involves the translocation of phosphatidylserine on the surface of cell membranes annexin V binding and propidium iodide uptake reveals various cellular states. After treatment with organotin(IV) compounds the cells could be categorized into populations vital cells (annexin V /P ), early apoptotic cells (annexin V /P ), late apoptotic cells (annexin V /P ), and necrotic cells (annexin V /P" ). Cells are observed with a fluorescence microscope and it is possible to observe translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer one and to see a green stain for annexin V FLUOS bound to PS, and a red stain for propidium iodide. 

Sioux and Teissie [203] loaded propidium iodide in 70% leukocytes in whole blood using the dielectric breakdown method. The entrapped drug showed a half-life of longer than 4 hours at 4 and 37°C. When compared with the nonpulsed cells, leukocytes loaded with the drug showed 10 times more accumulation in the inflammation area than in control areas. 

Apoptosis assay. ECRF24 or A2780 cells were seeded on 6-well plates (2 X 105 cells/ well) and grown 24 hours in complete medium before treatment. Compounds 1-3 were freshly dissolved in DMSO, diluted in complete medium and added to the cells at the final concentrations indicated in Table 2. After incubation for 72 h apoptosis was measured by flow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with propidium iodide (PI) as described previously10. Briefly, cells were harvested and subsequently fixed in 70% ethanol at —20°C. After 2 h the cells were re-suspended in DNA extraction buffer (45 mM Na2HP04, 2.5 mM citric acid, and 1% Triton X-100, pH 7.4) for 20 min at 37°C. PI was added to a final concentration of 20 pg/mL and log scale red fluorescence was analyzed on a FACSCalibur (BD Biosciences, NJ, U.S.). 

For cell cycle analysis, HOS cells (1 x 106) were treated with the indicated concentrations (see Table 13.2) of MTX or MTX-LDH for 20h, harvested by trypsin treatment, washed with PBS, and then fixed with cold 70 % ethanol on ice overnight. The fixed cells were incubated with propidium iodide and flow cytometric measurement was carried out. Over 20 h, an increase in the number of cells in the Gl phase resulted from MTX and MTX-LDH treatment compared to untreated cells, indicating arrest at the Gl/S boundary. It is worth noting that the inhibition of the Gl/S transition was more evident in the cells treated with MTX-LDH than in those treated with free MTX (85.59% versus 66.62% at 320pM/ml). This is consistent with... 

Mounting mount sections in aqueous medium or balsam for brightfield microscopy or in anti-fade medium for fluorescence microscopy (see Sect. 3.2.2). Notes. A11 incubations are at room temperature unless otherwise noted. Nuclear dyes (DAPI, Hoechst 33342 and Propidium Iodide) supplied as lyophilized solids are usually reconstituted in methanol. The stock solutions (5 mg/ml) are stable for many years when stored frozen at <—20°C and... 

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 ability of some fluorescent dyes to bind DNA quantitatively is exploited in flow cytometry to determine the DNA content of a cell. Dyes such as propidium iodide that bind double-stranded DNA stoichiometrically can be used for the purpose. The intensity of red fluorescence is directly related to the amount of DNA bound by propidium iodide. By comparing the fluorescence intensity of the test specimen and, in turn, its DNA content to the fluorescence intensity of specimens containing normal diploid amounts of DNA, a DNA histogram can be generated. By computing a DNA index, which is the ratio of DNA content of a test specimen to the DNA content of a specimen containing a normal diploid population, information related to the presence of an aneuploid tumor population can be obtained. The DNA index of 1 would imply that the DNA in the test specimen is from a normal diploid population (2N DNA), whereas the DNA index of an aneuploid population will be greater or less than 1. Thus, the DNA index of a tetraploid (4N DNA) would be 2. 

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

Cell cycle dynamics are closely connected to cell growth and to the mechanism of controlling cell proliferation. The cell cycle can be defined as an ordered set of biochemical events resulting in cell division. The sequence of these events is divided into four phases the Gi phase, followed by the S phase (DNA synthesis), G2 phase and the M phase. For determining percentage of cells at different phases of the cell cycle, cells must be stained for DNA content with propidium iodide (PI) [20, 21], Based on the amount of DNA content by PI, the fraction of cells in a specific phase can be determined [22] from whole population using dedicate software (e.g, Modfit or Flowjo ) or, more precise, by using 2D-dot plot BrdU incorporation [23],... 

Lecoeur, H., Prevost, M.C. and Gougeon, M.L., 2001, Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane A reconsideration ofthe specificity of the annexin V/propidium iodide assay. Cytometry, 44 65-72. 

Figure 4. SHIP-/- splenic B cells are resistant to BCR mediated induction of cell death. B cells were treated with anti-Ig(Fab)2 and surviving cells were detected after 12 h by propidium iodide staining. Reproduced with permission from Brauweiler et al., 2000, J. Exp. Med. 191. Rockefeller Univ. Press.

Figure 5. Induction of apoptosis in PC3 cells by PTEN. Cells were transplanted with empty vector (control) or PTEN, serum starved for 48h, and stained with annexin-FITC and SOpg/ml propidium iodide. Histogram shown is annexin-FITC staining of propidium iodide-negative cells. Reproduced with permission from Persad et al., 2000, Proc. Natl. Acad. Set. US A, 97. National Academy of Sciences, USA.

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),... 

Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139 271-279. 

Figure 7. Talin-containing streak-like focal adhesion plaques (A) and beta-actin cytoskeleton (B) in vascular smooth muscle cells in cultures derived from the rat aorta. Immunofluorescence staining, the nuclei couterstained with propidium iodide (A) or Hoechst 33342 (B). Microscope Olympus IX 50, digital camera DP 70, obj. 100, bar=20 pm.

Figure 27. Human osteoblast-like MG 63 cells in cultures on porous (A) or fibrous (B) poly(L-lactide-co-glycolide) scaffolds. A A summarizing picture of horizontal optical sections. The depth of cell ingrowth into the pores (average pore diameter of 400-600 mm) is indicated by spectral colors (blue 0-60 mm, green 80-160 mm, yellow 180-220 mm, orange 240-300 mm, red 320-400 mm, violet 420-480 mm). Day 14 after seeding, cells stained with propidium iodide. B cells grown for 4 days in static culture followed by 2 days in dynamic perfusion cell culture system. Cell membrane stained with Texas Red C2-maleimide and the nuclei counterstained with Hoechst 33342. Leica TCS SP2 confocal microscope, objective 5x (A) or lOx (B) [37].

Concentration of counterstain may have to be adjusted depending on the tissue being stained. Overstaining by propidium iodide may result in difficulty in observing the fluorescein label. 

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