Measurements with fluorescent dyes

Measurement with fluorescent dyes

Measurement with fluorescent dyes
Image Credit: PD Dr. Friederike Stumpff

The expression of proteins or their interaction with pharmacological substances or feed additives can change the intracellular concentration of ions, for instance the pH value or the intracellular calcium concentration. These processes can be monitored by loading the cells with appropriate dyes. To measure  intracellular Ca²⁺, the dye Fura-2-AM is frequently used. This dye is uncharged and diffuses into the cell, where it is split by cellular enzymes so that the charged product accumulate in the cell. Upon binding Ca²⁺, the  dye changes color. This change in the emission spectrum can be measured and calibrated, so that the intracellular Ca²⁺ concentration can be monitored over time.  After application of an agonist with opening of a Ca²⁺ permeable ion channel an increase is expected. In a second step, the Ussing chamber can be used in order to investigate whether tissue from the gut that expresses this channel also reacts to the substance in question. Such experiments can be useful to develope compounds that improve the Ca²⁺ supply of animals.