Orca flash 4.0 camera

E12.5 spinal cord ventral horns were obtained as above and infected with ChR2-eYFP and jRCaMP1b-expressing viruses at the same concentrations as indicated above for ChR2 (see above). Three days after infection, cultures were optically illuminated with 470 nm light pulses using the optical light fiber system, and changes in the red fluorescence were recorded using a BrightLine^TM filter: excitation FF01-554/23-25, emission FF01-609/54-25 filter cube and a Hamamatsu Orca Flash 4.0 camera through an Olympus IX71 inverted microscope with a 20× objective. Time-lapse images (1024 × 1024 pixels) were collected at 20 fps and integrated in Fiji™ in a single TIFF stack for further analysis. Several regions of interest (ROIs, eYFP-, and mRuby-positive) were selected, and the fluorescence trace F(t) was normalized nF(t) for each ROI to correct for its background brightness level by computing nF(t) = (F(t)−F₀)/F₀ ≡ ΔF/F₀, where F₀ is the average amplitude of the background fluorescence signal at rest.

The contractility of the myotubes was also monitored using a Hamamatsu Orca Flash 4.0 camera through an Olympus IX71 inverted microscope with a 20× objective. Time-lapse images (1024 × 1024 pixels) were collected at 40 fps and integrated in Fiji™ in a single TIFF stack for further analysis. To estimate the range of displacement and therefore the contractility of the cells, samples were processed by Matlab™ 2017b running in a Dell workstation. Matlab™ libraries were kindly provided by Dr. Nathaliel Huesbch (Dept. Biomedical Engineering, Washington University in St. Louis) [40 (link)]. With the software, among other parameters, the displacement/contraction or the contraction speed of selected ROIs containing C2C12 cells in the same optical plane over time were plotted and the average displacement (AD) in each experiment calculated. In order to do this, for reference purposes, the pixel size, fps, and size of the picture (1024 × 1024) were always the same. However, for the supplementary videos, portions of the analyzed videos were selected to better illustrate the presented data. All experiments were performed in triplicate unless specified. Videos were mounted using Final Cut Pro^TM software (iMac OSX 10.14.5).

To assess the ability of these scaffolds to induce blood vessel formation, a tube formation angiogenesis assay was conducted. The experiment was conducted using an angiogenesis kit procured from Gibco, USA as per the manufacturer’s protocol. Briefly, human umbilical vein endothelial cells (HUVEC) were cultured in a 96-well plate at a seeding density of 2.5 × 10³ viable cells/cm² and supplemented with Gibco™ large vessel endothelial supplement (LVES). The cells were then incubated at 37 °C in a humidified atmosphere of 5% CO₂. On the day of the tube formation assay, Geltrex™ LDEV-free reduced growth factor basement membrane matrix was prepared by placing it into the desired wells. The basement membrane matrix was then seeded with 25,000 cells per cm². Finally, supernatants from the scaffold wells were transferred to the matrix wells for the study. Wells containing Gibco™ large vessel endothelial supplement were used as the positive control and wells with only basement membrane matrix were the negative control. The angiogenic potential was then observed. The brightfield images were taken after 18 h of incubation using an inverted fluorescence microscope (IX-83, Olympus, Center Valley, PA, USA) equipped with an ORCA-flash 4.0 camera.