Medium-retaining Petri dish insert to grow and image cultured cells
Lech Kiedrowski1, Alan Feinerman2
1Department of Biological Sciences, The University of Illinois at Chicago, Chicago, IL 60607, USA.
Journal of Neuroscience Methods
|November 15, 2017
Summary
A new Petri dish insert prevents artificial intracellular calcium ([Ca2+]i) elevations during cell superfusion experiments. This method ensures basal Ca2+ levels remain undisturbed, improving experimental accuracy for cell signaling studies.
Area of Science:
- Cell biology
- Neuroscience
- Biophysics
Background:
- Microscope chambers for monitoring intracellular calcium ([Ca2+]i) in superfused cells often induce artifactual Ca2+ elevations.
- These elevations occur due to medium removal and re-application during coverslip installation.
Purpose of the Study:
- To develop and validate a novel method for monitoring intracellular Ca2+ in cultured cells without artifactual elevations.
- To maintain cells submerged throughout the experimental procedure.
Main Methods:
- A Petri dish insert with a superfusion-optimized well was designed to retain medium upon removal.
- The insert was tested using primary murine cortical neurons, astrocytes, and HEK-293 cells.
- The impact of transient medium removal on intracellular Ca2+ was assessed.
Main Results:
- The novel method successfully prevented artifactual intracellular Ca2+ elevations.
- Transient medium removal induced a Ca2+ spike in all tested cell types.
- The spike originated from Ca2+ influx in neurons and intracellular stores in astrocytes and HEK-293 cells.
Conclusions:
- The developed Petri dish insert effectively circumvents artificial Ca2+ level disturbances.
- This technique enables accurate monitoring of basal intracellular Ca2+ levels in various cell types.
- The method enhances the reliability of Ca2+ signaling studies.
