Super fluor 20 0.75 na

DRG neurons were loaded at 37°C with 3 μM Fura-2AM (K_d=25 μM, λ_ex 340, 380 nm/λ_emi 512 nm) to follow changes in intracellular calcium ([Ca²⁺]_c) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM NaHEPES, pH 7.4, 5 mM glucose exactly as previously described [13 ]. Fluorescence imaging was performed with an inverted microscope, Nikon Eclipse TE2000-U, using objective Nikon Super Fluor 20× 0.75 NA and a Photometrics cooled CCD camera CoolSNAPHQ (Roper Scientific, Tucson, AZ) controlled by MetaFluor 6.3 software (Molecular Devices, Downingtown, PA). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340 ± 5 and 380 ± 7) were controlled by a Lambda 10-2 optical filter change (Sutter Instruments). Fluorescence was recorded through a 505 nm dichroic mirror at 535 ± 25 nm. To minimize photobleaching and phototoxicity, the images were taken every ~5 seconds during the time-course of the experiment using the minimal exposure time that provided acceptable image quality. The changes in [Ca²⁺]_c were monitored by following a ratio of F340/F380, calculated after subtracting the background from both channels.

Dorsal root ganglion neurons were loaded at 37°C with 3 μM Fura-2AM (K_d = 25 μM, λ_ex 340, 380 nm/λ_emi 512 nm) to follow changes in intracellular calcium ([Ca²⁺]_c) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM NaHEPES, pH 7.4, 5 mM glucose exactly as previously described.^{9 (link),39 ,40 (link)} Fluorescence imaging was performed with an inverted microscope, Nikon Eclipse TE2000-U, using an objective Nikon Super Fluor 20× 0.75 NA and a Photometrics-cooled CCD camera CoolSNAPHQ (Roper Scientific, Tucson, AZ) controlled by MetaFluor 6.3 software (Molecular Devices, Downingtown, PA). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340 ± 5 nm and 380 ± 7 nm) were controlled by a Lambda 10-2 optical filter change (Sutter Instruments). Fluorescence was recorded through a 505-nm dichroic mirror at 535 ± 25 nm. To minimize photobleaching and phototoxicity, images were taken every ~2.4 seconds during the time course of the experiment using the minimal exposure time that provided acceptable image quality. The changes in [Ca²⁺]_c were monitored by following a ratio of F340/F380, calculated after subtracting the background from both channels.