Mohammad I K Hamad1, Martin Krause2, Petra Wahle3. 1. AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr Universität Bochum, D-44780 Bochum, Germany. Electronic address: mohammad.hamad@rub.de. 2. Lehrstuhl für Allgemeine Zoologie und Neurobiologie, Ruhr Universität Bochum, D-44780 Bochum, Germany. 3. AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr Universität Bochum, D-44780 Bochum, Germany.
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. NEW METHOD: 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. COMPARISON WITH EXISTING METHOD(S): 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. CONCLUSION: The method is optimal for calcium imaging in slice cultures.
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. NEW METHOD: 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. COMPARISON WITH EXISTING METHOD(S): 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. CONCLUSION: The method is optimal for calcium imaging in slice cultures.
Authors: Mohammad I K Hamad; Petya Petrova; Solieman Daoud; Obada Rabaya; Abdalrahim Jbara; Nesrine Melliti; Jennifer Leifeld; Igor Jakovčevski; Gebhard Reiss; Joachim Herz; Eckart Förster Journal: Development Date: 2021-09-06 Impact factor: 6.862
Authors: Morven Cameron; Orsolya Kékesi; John W Morley; Jonathan Tapson; Paul P Breen; André van Schaik; Yossi Buskila Journal: PLoS One Date: 2016-05-16 Impact factor: 3.240
Authors: Mohammad I K Hamad; Abdalrahim Jbara; Obada Rabaya; Petya Petrova; Solieman Daoud; Nesrine Melliti; Maurice Meseke; David Lutz; Elisabeth Petrasch-Parwez; Jan Claudius Schwitalla; Melanie D Mark; Stefan Herlitze; Gebhard Reiss; Joachim Herz; Eckart Förster Journal: J Neurochem Date: 2020-03-13 Impact factor: 5.372