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Operetta high content imaging and analysis system

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The Operetta High Content Imaging and Analysis system is a high-performance microscope-based imaging platform designed for cellular and subcellular analysis. It provides automated, multiparametric, and quantitative image-based data acquisition and analysis.

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Lab products found in correlation

Harmony and columbus software, 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) Harmony software, by PerkinElmer (1 mentions) Acapella software, by PerkinElmer (1 mentions) Cellcarrier ultra plates, by PerkinElmer (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Ab7349, by Merck Group (1 mentions)

Close spelling variants of this product

Operetta High Content Imaging System (352 citations) Operetta (246 citations) Operetta system (29 citations) Operetta imaging system (22 citations) Operetta high content analysis system (21 citations) High Content Analysis System (17 citations) High-Content Imaging System (13 citations) Operetta high-content system (8 citations)

10 protocols using operetta high content imaging and analysis system

1

High-Throughput Oligodendrocyte Viability Assay

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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.
Cohn E.F., Clayton B.L., Madhavan M., Yacoub S., Federov Y., Paul-Friedman K., Shafer T.J, & Tesar P.J. (2023). Pervasive environmental chemicals impair oligodendrocyte development. bioRxiv.
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2

Quantifying Oligodendrocyte Differentiation

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During hit validation experiments, 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 1,200 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.
Allimuthu D., Hubler Z., Najm F.J., Tang H., Bederman I., Seibel W., Tesar P.J, & Adams D.J. (2019). Diverse chemical scaffolds enhance oligodendrocyte formation by inhibiting CYP51, TM7SF2, or EBP. Cell chemical biology, 26(4), 593-599.e4.
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3

High-Content Imaging of OPC Differentiation

Cited 3 times
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Following fixing and staining OPC phenotypic assays for MBP positivity, plates were imaged on the Operetta High-Content Imaging and Analysis system (PerkinElmer). A set of 6 fields were captured in each well of each 96-well plate (4 fields for 384-well plates) resulting in an average of 1600 cells (500 cells for 384-well plate) scored per well. Analysis was performed using PerkinElmer Harmony and Columbus software. First, live nuclei were identified using the DAPI channel with a traced nucleus area between 50–250 μm2 which excludes pyknotic nuclei.11 (link),15 (link),18 (link) The traced nucleus region was then expanded by 50% and examined for myelin basic protein (MBP) staining to identify mature oligodendrocyte nuclei.15 (link) Dividing the number of mature oligodendrocyte nuclei by the number of live cells identified yielded % MBP positive oligodendrocytes.
Sax J.L., Hershman S.N., Hubler Z., Allimuthu D., Elitt M.S., Bederman I, & Adams D.J. (2022). Enhancers of human and rodent oligodendrocyte formation predominantly induce cholesterol precursor accumulation. ACS chemical biology, 17(8), 2188-2200.
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4

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)
Hubler Z., Allimuthu D., Bederman I., Elitt M.S., Madhavan M., Allan K.C., Shick H.E., Garrison E., Karl M., Factor D.C., Nevin Z.S., Sax J.L., Thompson M.A., Fedorov Y., Jin J., Wilson W.K., Giera M., Bracher F., Miller R.H., Tesar P.J, & Adams D.J. (2018). Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination. Nature, 560(7718), 372-376.
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5

Oligodendrocyte Differentiation Assay

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OPCs were seeded on 384-well poly-D-lysine CellCarrier Ultra plates (6057500, PerkinElmer), compound was added, and oligodendrocyte differentiation was induced. Three days later plates were fixed with 4% paraformaldehyde (PFA), and immunostained using rat anti-MBP and goat anti-Sox10, followed by counterstaining with DAPI. Images were captured and quantified using the Operetta High Content Imaging and Analysis system (PerkinElmer), Harmony software (PerkinElmer), and Acapella software (PerkinElmer).
Elitt M.S., Shick H.E., Madhavan M., Allan K.C., Clayton B.L., Weng C., Miller T.E., Factor D.C., Barbar L., Nawash B.S., Nevin Z.S., Lager A.M., Li Y., Jin F., Adams D.J, & Tesar P.J. (2018). Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease. Stem Cell Reports, 11(3), 711-726.
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6

Oligodendrocyte Quantification via Imaging

Cited 1 time
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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)
Hubler Z., Allimuthu D., Bederman I., Elitt M.S., Madhavan M., Allan K.C., Shick H.E., Garrison E., Karl M., Factor D.C., Nevin Z.S., Sax J.L., Thompson M.A., Fedorov Y., Jin J., Wilson W.K., Giera M., Bracher F., Miller R.H., Tesar P.J, & Adams D.J. (2018). Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination. Nature, 560(7718), 372-376.
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7

Quantifying Oligodendrocyte Populations

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During hit validation experiments, 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 1,200 cells being scored per well. Analysis (PerkinElmer Harmony and Columbus software) began by identifying live nuclei stained by DAPI; that is, those traced nuclei that were between 55–250 μm2 in area which excludes pyknotic nuclei. 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.
Hubler Z., Friedrich R.M., Sax J., Allimuthu D., Gao F., Rivera-León A.M., Pleshinger M., Bederman I, & Adams D.J. (2021). Modulation of lanosterol synthase drives 24,25-epoxysterol synthesis and oligodendrocyte formation.. Cell chemical biology, 28(6), 866-875.e5.
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8

Quantifying Oligodendrocyte Differentiation

Cited 2 times
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Both 96-well and 384-well plates were imaged using the Operetta High Content imaging and analysis system (PerkinElmer). For 96-well and 384-well plates, 8 fields and 5-fields were captured from each well at 20x magnification respectively. Images were analyzed with PerkinElmer Harmony and Columbus software as described previously (Hubler et al., 2018 (link); Najm et al., 2015 (link)). In brief, nuclei of live cells were identified using a threshold for area of DAPI staining to exclude pyknotic nuclei or debris. To identify oligodendrocytes, each DAPI positive nucleus was expanded by 50% to determine potential intersection with staining of an oligodendrocyte marker (O4/O1/MBP) in a separate channel. Expanded nuclei that intersected O4/O1/MBP staining were scored as oligodendrocytes. Percentage of oligodendrocytes was then calculated by dividing the number of oligodendrocytes by total number of DAPI positive cells per image.
Allan K.C., Hu L.R., Scavuzzo M.A., Morton A.R., Gevorgyan A.S., Cohn E.F., Clayton B.L., Bederman I.R., Hung S., Bartels C.F., Madhavan M, & Tesar P.J. (2020). Non-Canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function. Cell stem cell, 28(2), 257-272.e11.
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9

High Content Imaging of Oligodendrocyte Maturation

Cited 1 time
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The Operetta High Content Imaging and Analysis system (PerkinElmer) was used for plate imaging.13,14,17 (link) Each well had confocal imaging of 6 distinct fields at 20× lens. Cells were analyzed by quantifying nuclei stained with DAPI. Live cells were analyzed by excluding nuclei whose area ranged outside of 55–250 μm2 as these are pyknotic and represent dead cells. Typically, around 1500 live cells were imaged per well. Regions traced to nuclei belonging to live cells were then expanded 3 μm beyond the live nucleus. In this perinuclear region, the intensity of the MBP stain was measured to identify mature oligodendrocytes. Positive and negative control wells on each plate were used to establish thresholds for MBP intensity that accurately identified mature oligodendrocytes.
Pleshinger M.J., Friedrich R.M., Hubler Z., Rivera-León A.M., Gao F., Yan D., Sax J.L., Srinivasan R., Bederman I., Shick H.E., Tesar P.J, & Adams D.J. (2021). Inhibition of SC4MOL and HSD17B7 shifts cellular sterol composition and promotes oligodendrocyte formation. RSC Chemical Biology, 3(1), 56-68.
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10

Oligodendrocyte Differentiation Assay

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A 96-well plate containing Mimetex aligned scaffold (microfiber plate, AMSBIO, AMS. TECL005, Electrospun poly-L-lactide Scaffold) was sterilized with 70% ethanol and washed with PBS before being coated with polyornithine and laminin. After laminin coating, 40,000 cells were plated in differentiation medium. After 48 h the media was replaced with fresh media containing small molecule treatments for 14 days. Microfibre inserts were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 10% donkey serum (v/v) in PBS for 60 min. Then stained for MBP (Abcam, ab7349; 1:100) and DAPI staining (Sigma; 5 μg/ml). Wells were incubated for 45 minutes with Alexa Fluor conjugated secondary antibodies (1:500) for detection and imaged on the Operetta High Content Imaging and Analysis system (PerkinElmer). The total microfiber area was calculated using bright filed imaging and a spot-finding function (area larger than 2 px2). The MBP+ pixel area within the defined microfiber area was then defined and the percentage of the total microfiber area calculated.
Hubler Z., Friedrich R.M., Sax J., Allimuthu D., Gao F., Rivera-León A.M., Pleshinger M., Bederman I, & Adams D.J. (2021). Modulation of lanosterol synthase drives 24,25-epoxysterol synthesis and oligodendrocyte formation.. Cell chemical biology, 28(6), 866-875.e5.
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