Kbg and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes

Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for noninvasive K+ flux measurements and recording of activities of the dominant K+ channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 μM) evoked rapid (peaking around 15 min) transient K+ efflux, which then gradually decreased. This transient K+ efflux occurred concurrently with the transient increase of the K+ background (Kbg) TWIK-related spinal cord K+ channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kv1.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of Kbg and Kv1.3 channels to the transient and posttransient STS-induced K+ efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking Kbg current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K+ efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

Optimal Processing Method to Obtain Four-color Confocal Fluorescent Images of the Cytoskeleton and Nucleus in Three-dimensional Chondrocyte Cultures

Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilization protocols without optimization of parameters. In this study, we have examined procedures using glutaraldehyde and paraformaldehyde as fixatives and Triton X-100 and Octyl-POE as permeabilizing detergents. A four-color fluorescence confocal method was developed to simultaneously image actin, tubulin, vimentin, and the nucleus. We found optimal preservation and morphology of the chondrocyte cytoskeleton after simultaneous fixation and permeabilization with glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.

Ultrastructural Changes in Keratinizing Epithelium Following Trypsinization, Epidermal Detachment and Apposition to Mesenchymes

The cellular effects of trypsinization, a commonly used method for separating epithelial from mesenchymal tissues, were examined with the electron microscope. Specimens were fixed after each step of the trypsinization process in 1 % OsO4 in double strength Tyrode solution and after 1-7 days of culture following reapposition of the epithelium to its own mesenchyme or collagen-rich 12-day embryonic quadriceps tendon. Controls consisted of specimens identically prepared from limb bud skin derived from the same site from 5-day normal embryos to hatching. A second series of controls were 5-day skin explants cultured for 1-7 days in vitro following identical preparation except for the omission of trypsin. Trypsin caused transient cytoplasmic protrusions (‘blebs’) on the deep aspect of the basal cells, cellular shrinkage and increase in lysosomes. The basal lamina was disrupted overlying the blebs and remained attached to the basal cells only between the blebs. Continuity of the basal lamina was restored within 24 h after the onset of culturing and developed precociously thereafter, regardless of whether it was reapposed to its own collagen-poor mesenchyme or the collagen-rich quadriceps tendon. In the latter, focal excessive basal lamina formation was observed. Epithelial cell differentiation proceeded equally precociously in trypsinized and non-trypsinized cultures. Disruption of the basal lamina by trypsinization did not produce detectable alteration in the synthesis of tonofilaments, desmosomes, granular reticulum or corpuscula cribriformia. Keratohyaline bodies were not found in any of the cultures and keratinized cells did not develop.

p-Toluenesulphonic Acid as a Fixative

p-toluenesulphonic acid in aqueous solution is introduced to histologists and recommended for fixation of the central nervous system by a three-step procedure: flushing the blood-vessels with a saline solution, filling the vessels with the fixative, and delaying the autopsy. With rats and guinea-pigs as test objects, a solution of at least 0.5 M gave excellent results, as evidenced by the minimum of cellular shrinkage, the absence of perivascular and perineuronal spaces, and the clarity of cellular membranes and basiphil material. The neurones, neuroglia, microglia, and blood-vessels were well defined when stained by conventional histological techniques. Cytological details became more prominent because the tissue had shrunk less than in routine preparations. The acid is non-volatile, colourless, pleasant to handle, and low in price.