0 avis
A Simple Bioluminescence-Based Method for Recording Ca 2+ Signaling in the Cytosol or Mitochondria of Neural Progenitor Cells from the Adult Zebrafish Brain
Archive ouverte
Edité par CCSD ; Nova Science Publishers, Inc. -
International audience. Neurospheres derived from the adult zebrafish brain are a convenient model for investigating the initial stages of neurogenesis. Neurospheres are composed of stem cells, which have the ability to either self-renew or differentiate into specialized cells (i.e., neurons and glial cells) if cultured in an appropriate medium. It has previously been suggested that Ca 2+ signaling in the cytosol and mitochondria might play some role in neurogenesis. Ca 2+ signals can be recorded using the non-toxic bioluminescent Ca 2+ reporter complex, holoaequorin. We have developed a novel electroporation protocol to load neurospheres with cDNA encoding either mitochondrial-targeted EGFPapoaequorin (MitGA) or the cytosolic-targeted EGFPapoaequorin (CytGA). We show that within ~24 h after electroporation, each of these apoaequorin proteins were expressed in ~20% of the neurosphere cells. In addition, after reconstitution of holoaequorin by incubation of the cells with its luminophore, coelenterazine, dynamic changes in [Ca 2+ ] in the cytosol and mitochondria were imaged using a custom-built electron multiplying charge coupled device (EMCCD)-based photon imaging microscope (PIM) system or measured using a photomultiplier tube (PMT)-based luminescence detection system, after activation of store-operated Ca 2+ entry (SOCE).
Consulter en ligne
Suggestions
Du même auteur
