PROPIDIUM IODIDE PI

CA Index Name Phenanthridinium, 3,8-diamino-5-[3-(die thy lmethylammonio)propyl]-6-phenyl-, iodide (1 2) 

8-Diamino-5-(diethylmethylaminopropyl)-6-phenyl-phenanthridinium diiodide Propidium diiodide Propidium iodide PI Merck Index Number Not listed Chemical/Dye Class Heterocycle Phenanthridine 

Molecular Formula C27H34I2N4 Molecular Weight 668.40 Physical Form Red powder or solid 

Imaging/Labeling Applications Nucleic acids acetylcholinesterase bacteria cells chromosomes fimgi liposomes leukocytes, megakaryocytes miCTOorganisms neurons nuclei sperms stem cells yeasts  

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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.). 

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],... 

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),... 

It is useful to resuspend the stained cell pellet in 1 mL PBSG with 500 ng/mL propidium iodide (PI) to detect dead cells by the inclusion of PI and its resultant red fluorescence on the flow cytometer. Red fluorescence (PI) vs light scatter is used to exclude dead cells before collecting green (FITC) emitting cells of the viable cell population. 

After intracellular and surface staining are complete, it is often possible to utilize a stain for nucleic acids, such as propidium iodide (PI), DAPI, mithramycin, or... 

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. 

Propidium iodide (PI) prepare a stock solution of 50 pg/ml in water. Protect from light and store at -i-4°C. 

The addition of ethidium bromide (EtBr) or propidium iodide (PI) allows the identification of DNA bands immediately after run on a transilluminator. Intercalation of the dyes into the double-stranded DNA yields fluorescence (EtBr excitation wavelength Aex 302 or 366 nm, emission wavelength Aem 590 nm PI Aex 530 nm, Aem 620 nm). 

Instead of EtBr use less harmfull fluorescent dyes, e.g., propidium iodide (PI) or SYBR Green (cf. Haugland RP (1996) Handbook of fluorescent probes and research chemicals, 6th ed.. Chap. 8, Molecular Prohes, Eugene, Oregon https //catalog.invitrogen.com)... 

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. 

Stock solution of propidium iodide (PI) at 50 pg/mL This is stable if stored refrigerated m the dark (see Note 1). 

Fig. 8.12. Fluorescein (FITC) histogram, propidium iodide (PI) histogram, and dual-color correlated contour plot of human keratinocytes cultured for 4 days, pulsed with BrdU, and then stained with FITC-anti-BrdU and PI. Data courtesy of Malcolm Reed.

Fig. 8.16. Cell cycle analysis of CD8-positive and CD8-negative cells. Lymphocytes were cultured with phytohemagglutinin, stained with FITC anti-CD8 monoclonal antibody, treated with saponin to permeabilize the outer membrane, and then stained with propidium iodide (PI) and RNase. Cells provided by Ian Brotherick.

Fig. 8.19. A flow karyotype (fluorescence histogram) of Chinese hamster chromosomes stained with propidium iodide (PI). The G-banded chromosomes from this particular aneuploid cell line are included for comparison with the histogram peaks. From Cram et al. (1988).

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. 

Propidium iodide (PI, Molecular Probes, Eugene, OR) used at final concentration of 0.05 pg/mL for cell permeability quantification. 

Dextran conjugated to fluorescein-5-isothiocyanate (dextran-FITC, m.w. 20-2000 kDa, 0.009 mol FITC per mole glucose) is used at a final concentration of 0.2 iM. Bovine serum albumin conjugated to fluorescein-5-isothiocyanate (BSA-FITC) containing 12 mol FITC per mole albumin (m.w. 66 kDa) is applied at 1-10 iMconcentration. Dye-conjugated probes are dialyzed before use against the exposure medium. Propidium iodide (PI) is used at a final concentration... 

Add 8 g of cesium chloride to the 8 ml of supernatant and 0.6 ml of 2 mg/ml propidium iodide (PI). Some researchers have used ethi-dium bromide, but we recommend PI for maximum separation. Mix by inverting the tube, with Parafilm over top, until the CsCl has gone into solution. 

After intracellular and surface staining are complete, it is often possible to utilize a stain for nucleic acids, such as propidium iodide (PI), DAPI, mithramycin, or 7-aminoactinomycin D (7AAD) (see Chapter 26). The determination of which stain to use is dependent on the other fluorescent markers being used, spectral overlaps of the dyes, and the laser lines available for excitation (e.g., DAPI needs UV [345 nm] excitation light, whereas PI, 7AAD, and mithramycin use blue [488 nm] excitation light). 

The method outlined here uses a modification of the Hedley technique (9,10) to prepare nuclear suspensions from the paraffin-embedded tissue samples. Microtome sections are dewaxed, hydrated, and incubated in pepsin with intermittent vortexing and mechanical disruption to release the nuclei. After completion of the tissue digestion, the nuclei are either suspended in 70% ethanol for storage or stained with propidium iodide (PI) for FCM analysis. There are three alternative techniques for preparation of the nuclei on microslides, in tea bags, or in test tubes. 

Massive cell death in the experiments in vitro can be induced by high (0.5-3.0 mM) concentrations of kainate or NMD A. After exposure of neurons to such drugs the amount of cells being stained with propidium iodide, PI (so being dead) is proportional to the ROS... 

Propidium iodide (PI) staining solution PBS + 5 pg/mL PI (Molecular Probes, Eugene, OK) + 200 pg/mL DNase-free RNase (Sigma) make up fresh immediately before use. The RNase can be made as a stock of 20 mg/mL in H20 and stored in aliquots at -20°C. 

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