In an effort to help reduce the costs of fluorescence microscopy and expand the use of this valuable technique, we developed a low-cost platform capable of visualising and analysing the spatio-temporal dynamics of intracellular Ca 2+ signalling in astrocytes. The created platform, consisting of a specially adapted fluorescence microscope and a data analysis procedure performed with Imagej Fiji software and custom scripts, allowed us to detect relative changes of intracellular Ca 2+ ions in astrocytes. To demonstrate the usefulness of the workflow, we applied the methodology to several in vitro astrocyte preparations, specifically immortalised human astrocyte cells and wild-type mouse cells. To demonstrate the reliability of the procedure, analyses were conducted by stimulating astrocyte activity with the agonist dihydroxyphenylglycine (DHPG), alone or in the presence of the antagonist 2-methyl-6-phenylethyl-pyridine (MPEP).
Keyphrases
- low cost
- data analysis
- single molecule
- induced apoptosis
- cell cycle arrest
- high throughput
- wild type
- endothelial cells
- endoplasmic reticulum stress
- high resolution
- signaling pathway
- protein kinase
- cell death
- oxidative stress
- quantum dots
- optical coherence tomography
- electronic health record
- single cell
- cell proliferation