Application suite x las x version 3

Brain organoids (day 86 in culture) were loaded with 5 µM Fluo-4 AM (Thermo Fisher) and 0.2% Pluronic™ F-127 (Thermo Fisher) in calcium imaging buffer (130 mM NaCl, 4.7 mM KCl, 1 mM MgSO₄, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 20 mM Hepes and 5 mM glucose, pH 7.4) for 1 h in a cell culture incubator. Brain organoids were washed and transferred to 8-well µ-slides (ibidi, Germany) in 150 µl calcium imaging buffer and weighed down to prevent from moving. Fluorescence signals were detected every second for 141 s using a Leica TCS SPE laser scanning microscope equipped with a 20x objective, HC PL APO CS2 20x/0.75 DRY UV. Images were acquired with Leica Application Suite X (LAS X) Version 3.5.5. After 20 s of baseline measurement, 100 µl of a glutamate stock (250 μM; Sigma-Aldrich) was manually added to reach a final concentration of 100 µM in the well. Regions of interest (ROIs) in time series images were selected and analyzed using ImageJ (Schneider et al., 2012 (link)) and the change in fluorescence signals (ΔF/F) was calculated by: [F (signal at given time point) - F (mean of baseline signal)]/F (mean of baseline signal). Values above 0.2 ΔF/F were interpreted as a response to glutamate.

24 h before transfection, 5 × 10⁴ HEK293T cells were plated in each well of a 24 well plate (μ-Plate 24 Well Black ID 14 mm, 82426; ibidi). The cells were transiently transfected with N-terminal YFP-fused NF2 WT and mutant proteins using polyethylenimine (PEI, 1 mg/ml, 9002-98-6; Alfa Aesar) at 1:5 ratio of pDNA:transfection reagent. After 7 h, the medium was gently replenished. 24 h post-transfection, the cells were washed with PBS, followed by staining of the nuclei with Hoechst 33342 (20 mM, 12.3 mg/ml, 62249; Thermo Fisher Scientific) at a final concentration of 1 μg/ml according to the manufacturer’s instructions. Fluorescence microscope images were acquired by using a STELLARIS 5 Cryo Confocal Light Microscope (Leica) equipped with a HC PL APO 63x/1,40 OIL CS2 objective. For excitation, a 405 nm (Hoechst 33342) and a 514 nm (EYFP signal) laser were used. The emission range for the channels was set to 420 nm—505 nm (Hoechst 33342), and 545 nm—625 nm (EYFP signal). Image analysis was performed with the Leica Application Suite X (LAS X, version 3.5.5.) software package.