Fluorescence acridine orange

Another morphological assay of apoptosis is done with acridine orange, a nuclear staining that reveals chromatin condensation under light and fluorescent microscope. 

Acridine orange changes its fluorescence color from pale yellow-green to yellow in a specific pH range (pH 8-10) [1, 4]. 

Acridine orange, pH-dependent change of fluorescence color 91 Activation of the layer 124ff. N-Acylglycine conjugates 176 ADB = 2-amino-2, 5-dichlorobenzophe-none 227... 

It had been found that if bacteria are stained with acridine orange and examined under fluorescent microscopy, viable, as dishnct from dead, cells fluoresce with an orange-led hue. This basic observation has been adapted to an ingenious method of determining bacterial content and may be completed within 1 hour. 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

The pH indicator shows the acid or basic properties of sample molecnles. Commonly used for acid indicating are solutions of bromocresol green (20 mg dissolved in 10 ml of ethanol combined with 1 ml of 0.1-molar aqueous NaOH) or bromophenol blue (20 mg dissolved in 10 ml of ethanol, pH-adjusted with 0.1-molar NaOH or 0.2% aqneous citric acid). In the presence of acids, 2,6-dichloroindophenol (40 mg dissolved in 100 ml of ethanol) changes the color from blue to red. The fluorescent dye acridine orange (20 mg dissolved in 100 ml of ethanol) changes pH-dependently the color of its flnorescence from yellow-green to yellow. 

Using specific fluorescent transport substrates, inhibitors and kinetic analysis Using fluorescent xenobiotics or fluorescent analogue of xenobiotics Using special fluorescent xenobiotics or fluorescent analogues of xenobiotics Vital tests with acridine orange or neutral red Metachromatic fluorescence of intercalated or bound acridine orange, 590/530 nm microfluorometry... 

Fig. 10.46. Principles of optodes using a neutral carrier (e.g. valinomycin for K+) coupled to an amphiphilic fluorophore (A) an amphiphilic pH indicator (Seiler K. et al. (1989) Anal. Sci. 5, 557). (B) a fluorescent cationic surfactant (e.g. dodecyl acridine orange) (Kawabata Y. et al. (1990) Anal. Chem. 62, 2054).

The sensor reported by Shirai(69) used a natural carboxylic polyether antibiotic (Aem = 481 nm) for the detection of magnesium and calcium. Detection limits of I0 5 and KT4 M, respectively, were reported but, interference from other metals was difficult to overcome. Ishibashi(69) used a bulkier hexadecyl-acridine orange dye (Xem = 525 nm) plasticized in a PVC membrane for the fluorescent detection of ammonium ions. Signal interference due to superfluous ions and poor detection limits of KT5 M restricted the use of the probe. 

Some substituents tend to enhance fluorescence. They are known as fluorochromes in the same sense as auxochromes. In general, these are electron donors such as — OH, NH2, etc. which enhance the transition probability or intensity of colour, e.g. acridine and acridine orange. 

Comminution also may be used to examine the stability of dispersed phases such as oil droplets. Depending on the viscosity of the product one simply mixes it or breaks it up in a solvent (usually water but, for example, use fresh soybean oil for chocolate), a buffer or the appropriate dyes (below). For instance, we mix easily dispersible foods (cream cheese, ice cream mix or tablespreads) with dyes on slides in a ratio of about 1 1 before examination. Where the dye is a diachrome (that is, highly colored) or is fluorescent in the absence of the substrate (for example, Acridine Orange) some attempt must be made to remove excess, uncomplexed dye molecules which might confound the interpretation. This can be done by reduction of the dye concentration or by making the preparation thinner. The advantage of these simple techniques is that a battery of microchemical tests to identify protein, lipid and carbohydrate can be completed on multiple samples in a very short time period. 

Acridine Orange is a fluorescent marker that will reveal negatively charged groups. A 0.01 to 0.0001%W/V aqueous solution is added to the specimen - the concentration used depends on the difficulties posed by the background fluorescence of the dye. That is, the effective... 

Fig. 8.14. DNA (green fluorescence) versus RNA (red fluorescence) content of human leukemic cells stained with acridine orange. From Darzynkiewicz and Traganos (1990).

This acridine orange technique has been taken one step further by Darzynkiewicz s group. Because acridine orange fluoresces red when bound to single-stranded nucleic acid, but green with double-stranded nucleic acid, acridine orange can be used under mildly denaturing... 

Fig. 8.15. LI210 cells treated with RNase and acid and then stained with acridine orange reveal that DNA in mitotic cells is extensively unwound and exhibits increased red and decreased green fluorescence relative to DNA from cells in other phases of the cell cycle. From Darzynkiewicz (1990).

Probe Probe is a general term used, in flow cytometry, to refer to any chemical that fluoresces when it reacts or complexes with a specific class of molecules and therefore can be used to assay that molecule quantitatively. Propidium iodide and acridine orange are probes for nucleic acid because they complex specifically with nucleic acids and fluoresce brightly when they have... 

P 82] Dilution-type mixing was accomplished with the fluorescent dyes acridine orange (0.01% solution in 20 mM in TE buffer see below) or trypan blue (prepared in 0.85% saline) contacted with buffer solution (TE buffer 10 mmol f4 Tris-HCl, pH 7.4, 1 mmol 1 1 EDTA, pH 8.0) [164]. Images were taken by a laser scanning confocal microscope. Profiling data analysis was employed along detection lines. 

Figure 5. Dark-field fluorescence photomicrograph of 3T3 murine fibroblasts vitally stained with acridine orange a) treated with W. floribunda agglutinin (75 Hg/ml) in Eagle s Minimum Essential Medium with Hanks salts for 48 hr b)...

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