Orca flash 4.0 v2 c11440 22c camera

Calcium imaging was performed on DIV7 motoneurons plated on PORN/laminin-221/211 coated 35 mm high µ-dishes (81156; Ibidi). Cells were incubated with 5 µM Oregon Green 488 BAPTA-1, AM (O6807; Invitrogen, in Pluronic F-127; Thermo Fisher Scientific) or Cal-590 AM (ABD-20511; Biomol, in Pluronic F-127; Thermo Fisher Scientific) diluted in calcium imaging buffer (135 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 1 mM CaCl₂, 6 mM KCl, 5.5 mM Glucose, pH 7.4) for 15 min at 37°C. Upon three washing steps, cells were covered with calcium imaging buffer and placed into Tokai Hit stage incubator for imaging at 37°C with constant 5% CO₂ supply. Calcium imaging was performed at the Nikon’s Eclipse TE2000 inverted epifluorescence microscope equipped with a plan APO VC 60×/1.4 NA objective, a perfect focus system and NIS-Elements AR 4.40.00 software. A fluorescence LED light source was used for excitation at 470 nm (for Oregon Green) or 590 nm (for Cal-590 AM). 16-bit, 1,024 × 1,022-pixel pictures (2 × 2 binning) were taken at a frequency of 2 Hz over 5 min (exposure time 100 ms) at the ORCA Flash 4.0 V2 C11440-22C camera (Hamamatsu Photonics). The resulting 601 frame videos were analyzed with Fiji. Growth cones were defined as region of interest and a dynamic Z-axis profile, normalized against the average of the first 20 frames (F/F0), was plotted. Calcium spikes were counted using the BAR Plugin.

TrkB-GFP live-cell imaging was performed on motoneurons co-transduced with LV-TrkB-GFP and LV-mCh or LV-hPLS3 respectively, plated on PORN/laminin-221/211 coated 35 mm high µ-dishes (81156; Ibidi). ON DIV7, motoneurons were washed with pre-warmed Tyrode‘s solution (125 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 30 mM glucose, 25 mM HEPES, pH 7.4) and incubated with 100 µM 8-(4- Chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT-cAMP) containing Tyrode‘s solution for recording. Imaging was performed at the Nikon’s Eclipse TE2000 inverted epifluorescence microscope equipped with a plan APO VC 60×/1.4 NA objective, a perfect focus system and NIS-Elements AR 4.40.00 software. A fluorescence LED light source was used for excitation at 470 nm. 16-bit, 1,024 × 1,022-pixel pictures (2 × 2 binning) were acquired every ∼ 2.5 s over a time period of 10 min (exposure time 100 ms) using the ORCA Flash 4.0 V2 C11440-22C camera (Hamamatsu Photonics). The resulting videos were analyzed with Fiji.