Innocuousness and intracellular distribution of PKH67: a fluorescent probe for cell proliferation assessment.

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.

Methodological considerations on Feulgen-staining applied to cells in primoculture: the model of osteoarthritic synovial cells.

Human synovial cells in primoculture are an interesting model for the study of articular joint diseases and anti-rheumatic drugs. Based on results obtained by image cytometry of Feulgen-stained nuclei, we describe the heterogeneity of synovial cell populations and their progression during culture time in primoculture. Using the hydrolysis properties of the Feulgen reaction and their variations dependent on fixatives, we demonstrate the high acid-lability of the condensed chromatin observed in short term cultured nuclei compared to the acid-resistance of decondensed chromatin in long term cultured nuclei; these variations being probably induced by modifications in the molecular supra-organisation of chromatin during the aging of a culture. Finally, due to the cellular heterogeneity of the biological model and its evolution during culture progression, technical compromises are proposed to obtain optimal Feulgen staining, using Böhm-Sprenger fixative and a 1 h hydrolysis by 6 M HCl at 20 degrees C.