Nuclear microscopy of single whole cultured cells: Preparation and analysis of human Chang liver cells

Abstract

Nuclear microscopy is a powerful tool for the measurement of elemental concentrations in single cells. Six methods involving the use of various fixing agents, rinsing agents and drying methods were tried in the preparation of cultured human Chang liver cells for nuclear microscopy 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 commonly used criteria for the ionic integrity of cells; K/Na ratios well above 1 indicates minimal perturbation of the intracellular ionic composition. Non-stimulated human Chang liver cells in a resting state are usually polygonal in shape and flattened in firm anchorage to the substrate, while dividing or stimulated cells appear rounded. Therefore the shapes of the cells can be used as an indicator of whether the cells are 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 stimuli cannot be observed independently. Of the six methods tested, chemical fixation, as expected, was considered non-ideal for the preparation of human cultured Chang liver cells. Ice-cold 150 mM sucrose was found to be the most suitable rinsing solution for the preparation of cultured 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 found to be suitable although membrane blotting followed by air-drying was preferred as excess rinsing solution can be very quickly removed during the blotting process. The K/Na ratios of cells on the same target holder but from different regions were found to be dependent on the local cell density. Cells which are locally dense-packed were found to have a much higher K/Na ratio than cells in a less dense region.