Detection of Viable and Non-Viable Cells in Connective Tissue Explants Using the Fixable Fluoroprobes 5-Chloromethylfluorescein Diacetate and Ethidium Homodimer-1

By halogenation of methylfluorescein-diacetate (MFDA) or eosin-diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)-excitation system as required by bimane-based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37 degrees C, and fluorescence is sensitive to pretreatment with diethylmaleate but not to buthionine sulfoximine. In a dual-laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.

The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

We report on the morphology and structure of single and multiple chondrons isolated from homogenized samples of fresh and fixed canine tibial cartilage. Phase contrast, Nomarski, and scanning electron microscopy observations show each chondron to be composed of a chondrocyte and its pericellular matrix enclosed within a "felt-like" pericellular capsule. The extraction of intact chondrons from cartilage homogenates confirms the structural validity of the chondron concept and emphasizes the intrinsic mechanical strength of the capsule. Frayed collagen fibers radiate from multiple chondron columns suggesting a shear-resistant, structural interrelationship between capsular components and type II collagen fibers. Future development of chondron extraction procedures could provide a unique model with which to study the structure, biochemistry, and function of articular cartilage chondrocytes and their pericellular microenvironment.