Softworx imaging software

To visualize Ca²⁺ levels, D3(NMP-like) cells or D8 NPs (200K cells/cm²) were differentiated as described in Fig. 1B and incubated in a mixture of Fluo3AM (Invitrogen; stock 1 mM in DMSO, delivered to cells 1 μM) at 37°C for 30 min, rinsed with neurobasal medium (Gibco) supplemented as appropriate for D3 or D8 and left to recover for 1 h. Fluo3AM was then excited at 488 nm and the fluorescence generated was imaged by Deltavision Core microscope system in a WeatherStation environmental chamber maintained at 37°C. The D3(NMP-like) and D8 NP medium was buffered with a 5% CO₂/95% air mix and maintained in a humid chamber. Images were acquired using an Olympus 20×1.30 NA objective using a Xenon light source and a CoolSnap HQ2 cooled CCD camera (Photometrics). Images were deconvolved and maximum intensity projections of z-stacks were made using SoftWorx imaging software (Applied Precision). To provide a positive control for response to calcium influx, D3(NMP-like) and D8 NP cells were incubated with A23187 (Sigma C7522) 10 μg/ml in 0.1% DMSO in neurobasal medium) at 37°C for 20 min, rinsed, incubated in with Fluo3AM for 30 min and then rinsed in neurobasal medium. The fluorescence generated was imaged as above. The raw data were then quantified using ImageJ plugin Heatmap Histogram. Data and statistical analyses are presented in Fig. S7 and its legend.

pCS2 +-CLIP170-mOrange transfected COS1 cells were plated on a glass cover slip-overlaid plastic plate at a density of 1150 cells/mm². Cells were allowed to grow for 24 h. The glass cover slip was mounted onto a metal cassette with DMEM in 10% FBS to prepare for live-cell imaging. CLIP170-mOrange comets were imaged in a temperature- and humidity-controlled environment using a DeltaVision Core fluorescence microscope with a 60 × U-APO objective. Images were taken at 2-s intervals for 1 min and deconvoluted with Applied Precision softWoRx imaging software. Comets were and quantified using the MTrackJ plugin software in ImageJ/Fiji.