The concentration jump method

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Abstract
Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; R m values and the partition coefficients P o/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye C D can be increased from C D=0 to C D>0 within a few seconds (concentration jump). Thus, the time t=0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, C D=const., are unambiguously defined. The concentration of the dye, C b, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence I b. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, I f≪I b. The intensity of the fluorescence I=I b was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t → ∞. In the linear range of I(t) the flow J o=(dI/dt)o of the dye into the cell depends strongly on the dye concentration and increases linearly with C D. The concentration range C D=10−9−10−5 M at 37° C was investigated. From the linear correlation between J o and C D it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t=0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJ o/dC D of the plot J o vs C D describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.