Fluorescent dyes tetramethylrhodamine

Fig. 4.3.3. El ongation of the primer hybridized to the 5 -biotinylated template requires polymerization along a homopolymeric (dA)sa stretch. In the sole presence of fluorescence-labeled deoxynucleoside triphosphates (dye = tetramethylrhodamine, TAMRA), this selective constraint forces the multiple successive...

In metabolic cytometry, cells are incubated with a substrate that is tagged with a highly fluorescent dye we prefer tetramethylrhodamine because of its excellent spectroscopic properties and its compatibility with the frequency-doubled neodymium YAG laser. This substrate is prepared at high concentration and undergoes chromatographic purification to eliminate fluorescent impurities. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

In the preparation of 15 nm core-shell fluorescent silica particles, Ow et al. (2004) reported that the naked core (2.2 nm) alone produced a fluorescence intensity of less than the free dye in solution, presumably due to dye quenching. However, upon addition of the outer silica shell around the core, the brightness of the particles increased to 30 times that of the free dye (using tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)). They speculate that shell may protect the core from solvent effects, as evidenced by a lack of spectral shift upon changing the solvent in which the particles are suspended. 

Mitochondrial toxicity may also lead to a decrease in the mitochondrial membrane potential which can be measured by various dyes, such as Tetramethylrhodamine methyl ester (TMRM), Rhodaminel23, JC-1 (Molecular Probes), and mitochondrial membrane potential indicator (m-MPI, Codex). These dyes specifically accumulate in the matrix of the mitochondria according to the Nernst equation, with an inverse proportion to the A Pmembrane [9]. Thus, upon loss of mitochondrial membrane potential, the fluorescence intensity will decrease, as shown for TMRM in Fig. 2e, f. Notably, TMRM, is not a ratio-metric dye, which means that additional controls for mitochondrial number/ mass may be required, as in contrast to the ratio-metric dyes JC-1 and m-MPI. 

Make microelectrodes—we use 1.2-mm diameter, thin-walled aluminosilicate glass with internal filament (A-M Systems, Everett, WA). Electrode tip should have a nice constant taper and be fine but not wispy. When back-filled with dextran and 1M potassium chloride, they should have a resistance of between 50 and 150MQ. In practice, the precise electrode resistance does not matter if the electrode penetrates a cell, records a stable membrane potential, and passes sufficient dye, then it is a good electrode (see Notes 7 and 8). Electrodes should first be back-fiUed with approx 0.5 0L of fluorescent dextran (lOOmg/mL in distilled water. Molecular Probes cat. no. D-3308 and D-3306 for tetramethylrhodamine and fluorescein fluorescence, respectively) and then with a little 1M potassium chloride. Don t worry about air bubbles. The capillary action of the internal filament will deal with them. 

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