Relaxation technique for kinetic measurements of dye accumulation at the mitochondria of HeLa cells in situ

✍ Scribed by I. Siedel; H.W. Zimmermann

• Book ID: 103994524
• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 965 KB
• Volume: 27
• Category: Article
• ISSN: 1011-1344
• DOI: 10.1016/1011-1344(94)07073-w

✦ Synopsis

The lipophilic cationic fluorescent dye ESI4 specifically stains the mitochondria in living cells. It was used to determine he kinetics of vital staining of mitochondria in single selected HeLa cells by quantitative microfluorometry in the concentration ange Co = 6 x 10 -8 to 4 x 10 -6 M. The experiments were performed using the concentration jump method. They were evaluated with a quantitative staining model which considers dye binding as well as dye release at mitochondrial binding sites. The dnetic equations of the model are discussed in terms of the relaxation technique. The predictions of the theory agree well ~-ith the experimental results. The staining model was used to determine the rate constant kl = 7.6 x 102 s-1 M-~ and k2 = 1.6 x 10 -3 -t for dye binding and dye release respectively, and the binding constant K = 4.8 x 105 M-1 of dye molecules at the mitochondria n HeLa cells (values according to relaxation experiments). The constants are discussed in detail. In addition, some experiments • ~'ere performed on dye accumulation under the influence of antimycin, nigericin and oligomycin.