Axis metric plotting tool

Approximately 60,000–80,000 wild-type control or ALS mutant MNs along with 15,000–20,000 human primary astrocytes in 10 μL differentiation medium were plated on each well of a poly-l-ornithine-coated CytoView MEA 48-well plate. Glia-MN co-culture was used to allow for better uniform dispersal of MNs throughout the cultures for improved global recording within the wells. After 1–3 h of allowing the cells to settle, additional 300 μL of the medium was added to each well and further incubated at 37°C, 5% CO₂. Neuronal activity was recorded using Maestro Pro MEA system and AxIS Navigator software (Axion Biosystems). To exclude the possibility of the MEA signals being artifacts, tetrodotoxin (TTX) was used as a negative control to block action potentials. Following baseline recording, vehicle (H₂O) and two different concentrations of TTX (final concentrations 2.5 and 25 nM) were added sequentially to the wells and the activity was recorded for 600 s after each treatment. Additionally, recordings were made on wells with only astrocytes plated to determine if astrocytes are contributing to activities. All the collected data were digitized and analyzed with NeuralMetric Tool and AxIS Metric Plotting Tool (Axion Biosystems).

For multi-electrodes array (MEA), after control or sPIBF-treated hHOs were loaded in the MEA device (Maestro Edge version; Axion BioSystems, GA, USA), raw waveforms representing the field potential (FP) of each group were recorded for 3 minutes in AxIS software. Three leading indicators of recorded FP (spike amplitude, beat period, and field potential duration) were analyzed using the Cardiac Analysis Tool and AxIS Metric Plotting Tool (Axion BioSystems)^{57 (link),58 (link)}.

For FP measurement, iPSC-CMs were seeded in the cavity containing electrodes of the Geltrex-coated CytoView 6-well MEA plates (Axion BioSystems, Atlanta, GA, USA). Around 300,000 iPSC-CMs were resuspended in 20 µL cardio digestion medium and seeded in the electrode-containing cavity of the MEA plates. One hour later, an additional 1 mL of medium was added into each well. iPSC-CMs were kept in RPMI/B27 medium for one week before drug treatment. For every batch of experiment, at least two wells of iPSC-CMs from different plates were treated with the same condition to avoid plate variability. Spontaneous FP recordings were carried out using the Maestro Edge equipped with AxIS Navigator software (Axion BioSystems, Atlanta, GA, USA) with a sample rate of 12,500 Hz at 37 °C with 5% CO₂. From day 0 (right before treatment) to day 14 (last day for washout), FPs were recorded daily for all conditions used (Figure S1). Several key parameters including conduction velocity (CV), corrected FPD_C (corrected by Fridericia’s formula), and inter-beat interval were determined using AxIS Navigator, and further analyzed with AxIS Metric Plotting Tool (Axion BioSystems, Atlanta, GA, USA). Spontaneous beating frequency was defined as the reciprocal of averaged inter-beat interval. The mainstream CV values were averaged for one culture.