Medium-retaining Petri dish insert to grow and image cultured cells

摘要

Highlights ? For superfusion, coverslips with cultured cells must be removed from Petri dishes. ? This leads to air exposure and unintended [Ca 2+ ] i elevations. ? A method has been designed to avoid these unintended [Ca 2+ ] i elevations. ? Cells can be cultured in medium-retaining Petri dish inserts with glass bottom. ? When these inserts are removed from Petri dishes, the cells remain submerged. Abstract Background Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca 2+ concentration ([Ca 2+ ] i ) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca 2+ ] i elevations. New method To prevent these [Ca 2+ ] i elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times. Results These inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca 2+ ] i in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca 2+ ] i spike. While in neurons this spike was caused by a Ca 2+ influx, in astrocytes and HEK-293 cells, it was caused by a Ca 2+ release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca 2+ ] i ; in 24% of the astrocytes, the spike triggered [Ca 2+ ] i oscillations. However, prior to the spike, [Ca 2+ ] i was low and uniform in all these cells. Comparison with existing method(s) The new method avoids the artificially-induced [Ca 2+ ] i elevations that take place during the handling of glass coverslips with cultured cells. Conclusions The new method allows monitoring [Ca 2+ ] i without disturbing the basal [Ca 2+ ] i levels.