Fluorescein tetramethylrhodamine dextran

Fluorescein tetramethylrhodamine dextran (FRD) was used as a ratiometric probe, which provides a pH-independent signal (tetramethylrhodamine), to compensate for different organelle volumes, and a pH-dependent signal (fluorescein). The ratio of the pH-independent and pH-dependent fluorescence was expected to provide a quantitative measure of the pH. Acidic organelles (lysosomes and endosomes) were specifically labeled by FRD, because the cells endocytose small amounts of the extracellular medium, which is accumulated in endosomes and eventually lysosomes. CE was performed in a poly((V-acryloyl aminopropanol, AAP) modified capillary to reduce both adsorption at the capillary surface and the electroosmotic flow. LIF detection was performed off-column using a... 

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. 

Mix up a small ahquot of fluorescent dextran. Use Molecular Probes product numbers D-3308 and D-3306 for tetramethylrhodamine and fluorescein fluorescence, respectively. The author keeps a small frozen stock of dextran made up at a concentration of lOOmg/mL in distilled water from which he transfer a very small drop onto a Sylgard surface. As the water evaporates off, the dextran becomes sticky and is easily picked up on to the tip of a stainless-steel or tungsten micropin or onto the tips of watchmaker s forceps. Simply add more water to the drop of dextran if it dries out too much. 

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See also in sourсe #XX -- [ ]

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