A sealed preparation for long-term observations of cultured cells

INTRODUCTIONThe continuous long-term observation of cultured cells on the microscope has always been a technically demanding undertaking. This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO(2) control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. The preparation is an aluminum support slide with a square aperture cut in its center. The coverslip bearing the cells is attached to the top of the slide with a thin layer of silicone grease, and the bottom of the slide is similarly covered with a clean coverslip of the same size. The thickness of the slide is intended to coordinately maximize the volume of the medium while maintaining optical properties that allow Koehler illumination with standard condensers. The chamber is filled in equal parts with HEPES-buffered media containing fetal calf serum and a low-viscosity fluorocarbon oil. These oils have a high solubility for atmospheric gases. The inclusion of the oil in the preparations is intended to provide a source of oxygen and perhaps a sink for some of the CO(2) produced by the cells. Although the inclusion of fluorocarbon oil in the preparation may not be necessary for short-term (~24 hr) observations, particularly with cells that are sparsely plated, long-term cell viability is ensured when the oil is present.