Microscope stage incubator

Primary cortical neurons were infected 24 h after seeding with AAV9-Synapsin-GCaMP6f [60 (link)] (Watertown, MA, USA). In our cultures, the genetically encoded calcium indicators started to express 3–4 days after infection. Calcium changes in GCaMP6f-expressing neurons were recorded at 8, 11, 13, and 15 days in vitro (DIV) using an Olympus IX71 inverted microscope (Olympus, Hamburg, Germany), equipped with an ORCA-Flash 4.0 camera (Hamamatsu Photonics, Japan). During recording, the cells were maintained in a microscope stage incubator at 5% CO₂ and 37 °C (Okolab S.R.L., Italy). The same region of the culture was recorded throughout the days following the culture dish grid references. Images (1024 × 1024 pixels) were captured using a × 20 objective and 470 nm wavelength (CoolLED’s pE-300^white, Delta Optics, Madrid, Spain) every 100 ms for 8–10 min using the CellSens^TM software (Olympus) or the Micro-Manager Open Source Microscopy Software (https://micro-manager.org). Exposure levels and frequency were maintained between cultures and evaluation days. GCaMP6f activity was measured in four different identified squares of each culture dish during these 4 days.

Cells plated on 96-well glass-bottom plates were incubated at 37°C and 5% CO₂ by an Okolab microscope stage incubator with 96-well insert during all imaging experiments. Confocal microscopy was performed on a spinning disk (Yokogawa CSU-X1) confocal microscope with an Andor DU-897 EMCCD camera on a Nikon Eclipse Ti body using a 100x oil immersion Apo TIRF objective (NA 1.49). The following wavelength lasers were used to image the respective constructs: constructs with mGFP (488 nm), mCherry (561 nm), miRFP (640 nm). Fixed samples in the 53BP1 counting assay also used the 405 nm laser to detect nuclei stained with Hoechst (Thermo Fisher Scientific, H3570) or DAPI (Vectashield, H-2000-10).