title: Improving AM ester calcium dye loading efficiency.
abstract: BACKGROUND
  Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for calcium imaging. Pluronic F127 (PF127) as a surfactant and dimethyl sulfoxide (DMSO) as a solvent are used to dissolve the dyes, but concentrations vary between protocols. How these substances affect loading efficiency is not well characterized.
  
  
  METHODS
  We aimed to characterize dye loading in slice cultures. We determined minimum concentrations of surfactant, solvent and dye. The current study shows that the efficiency of AM dye loading depends on the initial stock concentration of PF127. Lowering the PF127 and DMSO concentrations can improve the loading efficiency.
  
  
  RESULTS
  Both, pluronic and DMSO are required for successful dye loading. However, dissolving the dyes in lower concentrations of PF127 yielded better staining efficiency. Moreover, lowering the DMSO concentration to ∼0.25% improves the efficiency. The strategy allows standard two-photon or confocal microscope monitoring of neuronal activity. The labeled cells display spontaneous and evoked calcium transients, and repetitive measurements for up to 24h after loading indicate that the method is not deleterious to neuronal function.
  
  
  CONCLUSIONS
  Dissolving the AM dyes in lower concentrations of PF127 combines the advantages of high loading efficiency, preserves cell viability and functional integrity, and allows repetitive measures over hours and days. Moreover, we found that the dye itself can be diluted to a final concentration of 1μM which reduces the experimental costs.
  
  
  CONCLUSIONS
  The method is optimal for calcium imaging in slice cultures.
authors.affiliation: AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr Universität Bochum, D-44780 Bochum, Germany. Electronic address: mohammad.hamad@rub.de.
authors.forename: Mohammad I K
authors.initials: MI
authors.lastname: Hamad
authors.affiliation: Lehrstuhl für Allgemeine Zoologie und Neurobiologie, Ruhr Universität Bochum, D-44780 Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Krause
authors.affiliation: AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr Universität Bochum, D-44780 Bochum, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Wahle
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008853:Microscopy / Q000379:methods*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D020442:Poloxamer / Q000737:chemistry; D051381:Rats; D012997:Solvents / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D046509:Tissue Culture Techniques; D014793:Visual Cortex / Q000502:physiology
chemical_list: D004396:Coloring Agents; D004952:Esters; D007211:Indoles; D012997:Solvents; D013501:Surface-Active Agents; D020442:Poloxamer; C059587:Indo-1 pentaacetoxymethyl ester; D002118:Calcium; D004121:Dimethyl Sulfoxide
keywords: AM ester dye; Calcium imaging; DMSO; Organotypic cultures; Osmolality; Pluronic PF127