fm1-43 and bis(1-3-dibutylbarbiturate)trimethine-oxonol

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Topics: Acridine Orange; Aniline Compounds; Barbiturates; Calcium; Cell Fractionation; Flow Cytometry; Fluorescent Dyes; Isoxazoles; Microscopy, Fluorescence; Neutral Red; Pyridinium Compounds; Quaternary Ammonium Compounds; Staining and Labeling; Vacuolar Proton-Translocating ATPases; Vacuoles; Vitis; Xanthenes; Yeasts

Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".

Topics: Animals; Barbiturates; Calcium; Egtazic Acid; Endocytosis; Exocytosis; Fluorescent Dyes; Isoxazoles; Membrane Potentials; Microscopy, Fluorescence; Motor Neurons; Muscles; Nerve Tissue; Oligochaeta; Potassium; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Vesicles