Basic NeuroscienceImproving AM ester calcium dye loading efficiency

- The efficiency of AM dye loading depends on the concentration of PF127.
- Both, PF127 and DMSO are required for successful dye loading.
- The labeled cells display spontaneous and evoked calcium transients.
- This method allows repetitive measurements for up to 24 h.
- This method is optimal for calcium imaging in slice cultures.

BackgroundCalcium 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 methodWe 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.ResultsBoth, 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 24 h 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 1 μM which reduces the experimental costs.ConclusionThe method is optimal for calcium imaging in slice cultures.