High-throughput Oligodendrocyte Quantification

The Operetta High Content Imaging and Analysis system was used to image all 96- and 384-well plates. For each well of the 96- and 384-well plates, 4 fields were captured at 20x magnification. The PerkinElmer Harmony and Columbus software was used to analyze images as described previously^{22 (link),50 (link),51 (link)}. In brief, nuclei from live cells were identified by DAPI positivity, using thresholding to exclude cell debris or pyknotic nuclei. A region outside of each DAPI-positive nucleus, expanded by 50%, was used to identify oligodendrocytes by positive staining for oligodendrocyte markers (O1 in primary screen and O4, O1, or MBP in kinetics experiments) within this region. Expanded DAPI-positive nuclei that overlapped with O4, O1, or MBP staining were classified as oligodendrocytes. Cell viability was calculated by dividing the number of DAPI- positive nuclei in an experimental well by the average number of DAPI-positive cells in the negative control wells. Oligodendrocyte percentage was calculated by dividing the number of oligodendrocytes by DAPI-positive cells and normalized to negative control wells.