Ix81 inverted fluorescence confocal microscope

Three-week-old neurons derived from BD and normal iPSCs were previously infected with a synapsin promoter-driven lentiviral vector expressing DsRed (Syn::DsRed). Cell cultures were washed twice with sterile Krebs HEPES Buffer and incubated with 3 μm Fluo 4-AM (Molecular Probes) in Krebs HEPES Buffer for 40 min at room temperature. Excess dye was removed by washing twice with Krebs HEPES Buffer, and cells were incubated for an additional 20 min to equilibrate the intracellular dye concentration and allow de-esterification. Time-lapse image sequences (×100 magnification) of 3,000 frames were acquired at 28 Hz with a region of 336 pixels × 256 pixels using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics) with a 488 nm (FITC (fluorescein isothiocyanate)) filter on an Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical). To assess changes in calcium signalling in response to perturbation of neuronal activity, tetrodotoxin (1 μm) was applied by bath application. Images were acquired with MetaMorph 7.7 software (MDS Analytical Technologies). Images were subsequently processed using ImageJ software and custom written routines in Matlab 7.2 software (Mathworks).

Neuronal networks derived from human iPSCs were transduced with lentivirus carrying the Syn::RFP reporter construct. Cell cultures were washed with Krebs HEPES Buffer (KHB) (10 mM HEPES, 4.2 mM NaHCO₃, 10 mM dextrose, 1.18 mM MGSO₄, 1.18 mM KH₂PO_4, 4.69 mM KCl, 118 mM NaCl, 1.29 mM NaCl₂; pH 7.3) and incubated with 2–5 μM Fluo-4AM (Molecular Probes/Invitrogen, Carlsbad, CA) in KHB for 40 min. 5,000 frames were acquired at 28 Hz with a region of 256 × 256 pixels (100x magnification), using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC) filter on an Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical, Japan). Images were acquired with MetaMorph 7.7 (MDS Analytical Technologies, Sunnyvale, CA), processed and analyzed using individual circular regions of interest (ROI) on ImageJ and Matlab 7.2 (Mathworks, Natick, MA). Syn::RFP+ neurons were selected after confirmation that calcium transients were blocked with 1 mM of tetrodotoxin (TTX). The amplitude of signals was presented as relative fluorescence changes (ΔF/F) after background subtraction. The threshold for calcium spikes was set at the 95^th percentile of the amplitude of all detected events.