Immunocytochemistry and fluorescence microscopy
All washes and incubation steps were performed in PBS with 0.01% w/v sodium azide (PBS-NaN3) unless otherwise stated. Cell cultures were fixed for 15?min at RT in 4% w/v paraformaldehyde (PFA) in PBS. Fixed cells were blocked and permeabilized with 0.1% Triton X-100 with 10% v/v goat serum (Vector Laboratories; UK) in PBS for 1?hour at RT. Primary and secondary antibody sources and dilutions are shown in Table 1. Primary antibody incubations were overnight at 4°?C, followed with PBS wash, secondary antibody incubations at RT for 1?hour followed with PBS wash, stained for 10?min at RT with DAPI or Hoechst-33342 (Life Technologies; UK) followed with PBS wash prior to mounting in Mowiol with 2.5% w/v DABCO. Omission of primary antibodies was used to verify specificity as control in all experiments. Photo-micrographs were taken with using Leica SP5 confocal or Leica DM inverted microscopes (Leica; UK).


Binarised images of MBP and IB4 were used to calculate fraction areas with ImageJ v1.48, and normalized to controls for each biological replicate. For Sholl analysis, GFAP images were binarised and branches manually traced using the NeuronJ plugin in ImageJ followed with Sholl Analysis v3.4.1 plugin in ImageJ55. Sholl parameters were 10?μm starting radius with 2.5?μm steps. Viability was calculated as cell numbers with DAPI and cell specific marker (NG2 or β-III-tubulin) minus those with PI, and values normalized to control conditions.

Calcium Imaging
Coverslips with OPCs were placed in either DMEM?+?SATO or MEMO?+?SOS with mitogens for ~24?hrs, prior to loading LED Candle Lights with 4?μM Fura2-AM (Life Technologies; UK) for 1?hour at 37?°C. Coverslips were placed on an Olympus IX71 microscope superfused with buffered Ringer’s solution containing the following (in mM) 124 NaCl, 2.5 KCl, 2 MgCl2, 1 NaH2PO4, 26 NaHCO3, 10 Glucose and 2.5 CaCl2 and bubbled with 95% O2, 5% CO2. Fluorescent images from 340?nm and 380?nm excitations were collected, on average from all experiments (n?=?10), for 83.9?±?1.54?minutes at an average of ~1 frame per second (0.78?±?0.02), with an exposure time of 200?ms. The emission of Fura2-AM is measured at 510?nm after excitation at 340?nm and 380?nm and the ratio of these emission intensities correlates with the calcium concentration within the cell.

Statistical Analysis
Numbers of experiments are indicated on bargraphs, data shown as mean?±?standard error of the mean (s.e.m.), and assumed to follow normal distribution. P values from Student’s two tailed unequal variance t-tests?<?0.05 were considered significant.

Additional Information
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