Improving AM ester calcium dye loading efficiency

Journal of Neuroscience Methods
Mohammad I K HamadPetra Wahle

Abstract

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. 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. 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. Dissolvi...Continue Reading

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Citations

Nov 14, 2017·Journal of Neural Engineering·Renaud SchuckSimon R Schultz
Feb 22, 2020·Journal of Neurochemistry·Mohammad I K HamadEckart Förster
Aug 18, 2020·Invertebrate Neuroscience : in·Paul D E WilliamsRichard J Martin
Nov 23, 2019·Scientific Reports·Nancy T MalintanDavid B Sattelle
Nov 17, 2020·Frontiers in Cellular Neuroscience·Ina GasterstädtPetra Wahle
Dec 8, 2020·Current Protocols in Neuroscience·Lakshmini BalachandarJorge Riera Diaz

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