NUCLEAR MICROSCOPY OF SINGLE WHOLE CULTURED-CELLS - PREPARATION AND ANALYSIS OF HUMAN CHANG LIVER-CELLS

Nuclear microscopy is a powerful tool for the measurement of elemental concentrations in single cells. Six methods involving the use of vari ous fixing agents, rinsing agents and drying methods were tried in the preparation of cultured human Chang liver cells for nuclear microscop y and the suitability of each method was evaluated by monitoring the K /Na ratios and shapes of individual cells. The K/Na ratio is a commonl y used criteria for the ionic integrity of cells; K/Na ratios well abo ve 1 indicates minimal perturbation of the intracellular ionic composi tion. Non-stimulated human Chang liver cells in a resting state are us ually polygonal in shape and flattened in firm anchorage to the substr ate, while dividing or stimulated cells appear rounded. Therefore the shapes of the cells can be used as an indicator of whether the cells a re in a resting or stimulated state. It is not desirable for cells to be in a stimulated state since then the effects of other external stim uli cannot be observed independently. Of the six methods tested, chemi cal fixation, as expected, was considered non-ideal for the preparatio n of human cultured Chang liver cells. Ice-cold 150 mM sucrose was fou nd to be the most suitable rinsing solution for the preparation of cul tured human Chang liver cells. Both freeze-drying and air-drying were used as drying methods and cells processed by either method were found to have K/Na ratios well above 1. Hence both drying methods were foun d to be suitable although membrane blotting followed by air-drying was preferred as excess rinsing solution can be very quickly removed duri ng the blotting process. The K/Na ratios of cells on the same target h older but from different regions were found to be dependent on the loc al cell density. Cells which are locally dense-packed were found to ha ve a much higher K/Na ratio than cells in a less dense region.