Pluronic f 127 detergent

DRG neurons were washed twice in basal solution containing (mM): 145 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, one sodium citrate, 10 HEPES, 10 D-glucose, pH 7.4, and incubated in the same solution containing 5 uM fura-2 with 0.05% Pluronic F-127 detergent (Life Technologies) for 1 hr at 37°C, 5% CO₂. Afterwards, cells were washed twice in same solution and placed on an inverted Nikon Ti-eclipse microscope with a Nikon Plan fluor 20x objective (0.45 N.A.). Fura-2 measurements were recorded at excitation wavelengths of 340 and 380 nm using EasyRatioPro (HORIBA Scientific). DRG neurons were depolarized with a solution in which NaCl was reduced to 110 mM and KCl increased to 40 mM.
Pancreatic β-cells were imaged with a similar protocol. Cells were maintained in a basal KRBH solution composed of (mM): 134 NaCl, 3.5 KCl, 1.2 KH₂PO₄, 0.5 MgSO₄, 1.5 CaCl₂, 5 NaHCO3, 10 HEPES, 2.8 D-glucose, pH 7.4. Stimulation solutions included either 16.8 mM glucose or 40 mM KCl, with NaCl concentrations adjusted accordingly to balance osmolarity with KRBH solution.

Hippocampal neurons were washed twice in basal solution containing (mM): 150 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 10 D-glucose, pH 7.4, and incubated in the same solution containing 2 μM fura-2 with 0.05% Pluronic F-127 detergent (Life Technologies) for 30 min at 37 °C, 5% CO₂. Cells were subsequently washed twice in the same solution and placed on an inverted Nikon Ti-eclipse microscope with a Nikon Plan fluor 20x objective (0.45 N.A.). Fura-2 measurements were recorded at excitation wavelengths of 340 and 380 nm using EasyRatioPro (HORIBA Scientific). Hippocampal neurons were depolarized with a solution in which NaCl was reduced to 65 mM and KCl increased to 90 mM.