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

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Abstract
The efficacy of connective tissue explants is difficult to determine, particularly where autopsy material is required for research or clinical applications. We report here an optimised protocol using 5-chloromethyl fluoresce in diacetate (CM FDA) and ethidium homodimer-1 to distinguish viable and non-viable cells in a range of connective tissue explants. Biopsies and explants of corneae, arteries, cartilage and skin were loaded with fluoroprobes for extended periods (≤ 24h) at low temperatures (4°C), fixed in paraformaldehyde, and processed using a variety of embedding, sectioning, autoradiographic, and immunohistochemical procedures. Detection of fluorescent green CM FDA and red ethidium homodimer was achieved using epi-illuminated light or dual channel confocal microscopy, and clearly differentiated live from dead cells throughout the explants. Furthermore, the intracellular distribution of CMFDA provided superior images of cell shape and morphology not previously available using conventional histochemical techniques. Adaptations of this protocol could prove valuable in a variety of research and clinical applications.