c.i.-fluorescent-brightening-agent-28 and diacetylfluorescein

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H

Topics: Benzenesulfonates; Blotting, Western; Catharanthus; Cell Fractionation; Enzyme Assays; Fluoresceins; Fluorescent Dyes; Hydrolysis; Microscopy, Fluorescence; Neutral Red; Optical Imaging; Osmotic Pressure; Plant Leaves; Plant Proteins; Plants, Medicinal; Protoplasts; Pyridinium Compounds; Quaternary Ammonium Compounds; Staining and Labeling; Vacuolar Proton-Translocating ATPases; Vacuoles

Cysts of Blastocystis ratti were produced in vitro by culturing the parasite in Iscove's modified Dulbecco's medium (IMDM) with increasing concentrations of horse serum. Yields up to 3 x 10(6) cysts/ml of culture medium were obtained after 72 h. Encystation efficiency was time, strain and inoculum size dependent. A viability of > 70% was determined by flow cytometry employing fluorescein diacetate and propidium iodide staining. The presence of chitin as a cyst wall component was demonstrated by Calcofluor White M2R staining with which cystic stages showed blue fluorescence. The changes in morphology during excystation were examined by transmission electron microscopy. The cyst enlarged in size and some vacuoles appeared within the condensed cytoplasm. The vacuoles were full of inclusions and small glycogen aggregates. Coalescence of the vacuoles led to central body formation. Glycogen deposits were prominent throughout the excystation process. Some cysts divided by binary fission before the completion of the excystation.

Topics: Animals; Benzenesulfonates; Blastocystis; Blastocystis Infections; Culture Media; Flow Cytometry; Fluoresceins; Microscopy, Electron; Microscopy, Fluorescence; Propidium; Rats; Rats, Sprague-Dawley; Rats, Wistar; Staining and Labeling

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.

Topics: Animals; Benzenesulfonates; Cell Line; Cell Survival; Edible Grain; Eukaryota; Flow Cytometry; Fluoresceins; Fluorescent Antibody Technique; Mice; Mice, Inbred BALB C; Propidium; Rats; Spleen