OPCs were seeded in 384-well plates (PerkinElmer, 6057500) pre-coated with poly-D-lysine and laminin (Sigma, L2020) at a density of 12,500 cell per well and allowed to attach for 1 hour at 37°C. Cell death inhibitors quinoline-Val-Asp-Difluorophenoxymethylketone (QVD-OPH) (Selleck, S7311), ferrostatin-1 (Selleck, S7243), and necrostatin-1 (Selleck, S8037), were added using a Janus automated workstation and 50 nL solid pin tool attachment in 8-point dose response (80 nM to 10 µM), and incubated for 1 hour at 37°C. Methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride was added to all wells at IC90 concentrations (approximately 100 nM), and oligodendrocytes were allowed to develop for 72 hours. Negative control wells contained only methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride. Positive control wells contained vehicle (DMSO). Cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, HP1–100Kit) and stained with and DAPI (1 μg/mL, Sigma, D8417). Imaging was performed with the Operetta High Content Imaging and Analysis system (PerkinElmer) and the PerkinElmer Harmony and Columbus software was used to quantify DAPI-positive nuclei.
Harmony and columbus software
Manufactured by PerkinElmer
Harmony and Columbus software are image analysis and data management solutions developed by PerkinElmer. Harmony software provides advanced image analysis and data management capabilities, while Columbus software offers a comprehensive platform for image-based screening and analysis. Both software solutions are designed to support various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Operetta high content imaging and analysis system, by PerkinElmer (5 mentions)
Necrostatin 1, by Selleck Chemicals (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
D8417, by Merck Group (1 mentions)
Poly d lysine and laminin, by Merck Group (1 mentions)
Ferrostatin 1, by Selleck Chemicals (1 mentions)
6 protocols using harmony and columbus software
1
High-Throughput Oligodendrocyte Viability Assay
2
Quantifying Oligodendrocyte Differentiation
3
Oligodendrocyte Quantification via Imaging
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Plates were imaged on the Operetta High Content Imaging and Analysis system (PerkinElmer) and a set of 6 fields captured from each well resulting in an average of 1200 cells being scored per well. Analysis (PerkinElmer Harmony and Columbus software) began by identifying intact nuclei stained by DAPI; that is, those traced nuclei that were larger than 300 μm2 in surface area. Each traced nucleus region was then expanded by 50% and cross-referenced with the mature myelin protein (MBP) stain to identify oligodendrocyte nuclei, and from this the percentage of oligodendrocytes was calculated. In some experiments, PLP1 staining was performed instead of MBP, or the total process length of MBP+ oligodendrocytes was calculated as previously described. 4 (link)
4
High-throughput Oligodendrocyte Quantification
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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 previously22 (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.
5
Oligodendrocyte Quantification via Imaging
6
Quantifying Oligodendrocyte Populations
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