phalloidine and fluorexon

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0-1000 V/cm for a selected duration in the range 10-1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.

Topics: Animals; Biological Transport; Cell Line, Tumor; Cell Membrane; Cell Survival; Electricity; Electrophoresis; Electroporation; Erythrocytes; Fibroblasts; Fluoresceins; Fluorescent Dyes; Humans; Male; Phalloidine; Rats

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.

Topics: Actins; Animals; Astrocytes; Biophysical Phenomena; Biophysics; Brain; Cells, Cultured; Fluoresceins; Microscopy, Fluorescence; Neuroglia; Phalloidine; Rats; Rats, Sprague-Dawley

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

Topics: Alloys; Biocompatible Materials; Blotting, Northern; Calcification, Physiologic; Calcium; Cell Adhesion; Chromium Alloys; Fluoresceins; Fluorescent Dyes; Humans; Osseointegration; Osteoblasts; Osteosarcoma; Phalloidine; Prostheses and Implants; Titanium; Tumor Cells, Cultured

Common dental implants are made of different grades of commercially pure titanium (cpTi) that are more than 99% similar in chemical composition. The objective of this in vitro study was to determine if human osteoblast-like cells, Saos-2, would respond differently when plated on disks of cpTi Grade 1 and Grade 4. Glass disks served as controls. In spite of identical preparation, the two grades of cpTi acquired different surface topographies, as illustrated by scanning electron micrographs and profilometry. Cell responses, such as adhesion, morphology, and collagen synthesis also differed on the two grades of cpTi. Between 4 and 24 h, the rate of cell attachment to Grade 1 differed significantly compared to cell attachment to Grade 4 and to glass. Rhodamine phalloidin fluorescence microscopy showed variations in the actin-based cytoskeleton between grades 1 and 4 cpTi in cell spreading, shape, and the organization of stress fibers. Immunofluorescent staining showed differential expression of vinculin, a focal adhesion protein, on the substrates. At 24 h, the percent of collagen synthesized was significantly more on Grade 1 than on Grade 4 and on glass. Alkaline phosphatase activity was similar on all substrates. The calcium content was significantly higher on Grade 1 than on Grade 4 and on glass at 24 h and at 4 weeks. Thus, commonly used cpTi induced differential morphologic and phenotypic changes in human osteoblast-like cells depending on the grade of the material.

Topics: Alkaline Phosphatase; Calcium; Cell Line; Collagen; DNA; Electron Probe Microanalysis; Fluoresceins; Fluorescent Antibody Technique; Fluorescent Dyes; Glass; Humans; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Osteoblasts; Phalloidine; Surface Properties; Titanium