Differentiated cells were fixed with 4% formaldehyde prior to staining with Hoechst (nuclei), BODIPY (lipid droplets), and Syto60 (cytosol) (Invitrogen). Images were taken per well with an automatic imaging system (Operetta, Perkin Elmer) and analyzed for lipid droplet content using the Operetta software.
Operetta
Manufactured by PerkinElmer
Sourced in United States, United Kingdom, Germany, Canada
The Operetta is a high-content imaging system designed for cell-based assays. It enables automated image acquisition, analysis, and data management for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Harmony software, by PerkinElmer (24 mentions)
Hoechst 33342, by Thermo Fisher Scientific (15 mentions)
Columbus software, by PerkinElmer (14 mentions)
Harmony 3, by PerkinElmer (9 mentions)
Hoechst, by Thermo Fisher Scientific (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Bodipy, by Thermo Fisher Scientific (6 mentions)
Harmony high content imaging and analysis software, by PerkinElmer (5 mentions)
Cellcarrier, by PerkinElmer (4 mentions)
Ab97959, by Abcam (4 mentions)
Nucblue, by Thermo Fisher Scientific (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Harmony analysis software, by PerkinElmer (3 mentions)
Acapella software, by PerkinElmer (3 mentions)
Columbus database, by PerkinElmer (3 mentions)
Tryptose phosphate broth, by Merck Group (3 mentions)
Cellcarrier 384 ultra, by PerkinElmer (3 mentions)
Non essential amino acids (neaa), by Merck Group (3 mentions)
Syto60, by Thermo Fisher Scientific (3 mentions)
246 protocols using operetta
1
High-Content Lipid Droplet Analysis
2
Analysis of Tau Phosphorylation and Neurodegeneration in Primary Neurons
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To detect endogenous tau phosphorylation in primary neurons, therneurons were fixed by 3.7% paraformaldehyde (PFA, Sigma) after 48 hrs of K18-wt and K18-P301L treatment. Then, the neurons were incubated in 0.1% triton-X in PBS for permeabilization. After washing with PBS, primary neurons underwent blocking step by 4% BSA for 1 hr followed with incubated with primary phospho-tau antibody Ser 199/202 (1:1000, abcam) overnight at 4 °C. Next day, primary neurons were stained with Alexa Fluor 488 or 633-conjugated secondary antibodies (1:1000, abcam). Images were obtained by the Operetta (PerkinElmer™). The fluorescence intensities were measured in 50 ~ 60 neuronal somas in 5 images from each PBS, K18-wt and K18-P301L-treated primary neurons. Quantification data was analyzed by F-test.
For neuronal degeneration analysis, primary neurons were stained with 250 nM NeuO, neuron selective probe22 . After 1 hr incubation with NeuO, images were obtained by the Operetta (PerkinElmer™,). The neurite length and the number of extremities were analyzed by Harmony 3.1 software (PerkinElmer™) (Fig. S9 ). Quantification data was analyzed by Student’s t-test with 95% significance level.
For neuronal degeneration analysis, primary neurons were stained with 250 nM NeuO, neuron selective probe22 . After 1 hr incubation with NeuO, images were obtained by the Operetta (PerkinElmer™,). The neurite length and the number of extremities were analyzed by Harmony 3.1 software (PerkinElmer™) (
3
Immunocytochemistry of EpCAM-sorted cells
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The EpCAM sorted subpopulation, parental PMC42-LA cells and the single cell-derived clones were seeded at a density of 10,000 cells/well in 48-well plates (Thermo Scientific NunclonTM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified phosphate-buffered saline (DPBS; pH 7.5). Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with the designated primary antibodies at 4 °C overnight. After rinsing in DPBS, cells were incubated for 2 h at room temperature in the dark on a gentle rotary shaker with appropriate fluorescence-conjugated secondary antibody (Supplementary Table S2 ) and with diamidino phenyl indole (DAPI) as a nuclear stain (diluted to a final concentration of 1 µg/mL). The plates were then washed thrice with DPBS and images captured on a high-content imaging platform (Cytell Cell Imaging System (GE Healthcare, Buckinghamshire, UK), IN Cell Analyzer 6000 (GE Healthcare, Buckinghamshire, UK) or PerkinElmer Operetta® (PerkinElmer, Waltham, MA, USA) as indicated) with approximately 9 fields of view taken per well. Images were analyzed and merged using the respective software; IN Cell Investigator software v1.0 (GE Healthcare) or Harmony® v4.8 (PerkinElmer).
4
Directional Cell Migration Assay
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Cells were seeded into the reservoirs of 2-well cell culture silicon inserts at a density of 3 × 104 cells/well (Ibidi, Martinsried, Germany). The inserts were designed to ensure directional cell migration, with a defined cell-free gap of 500 μm. Upon cellular attachment, the medium was replaced with serum-reduced medium for 24 h to suppress cell division. After nuclear staining with Hoechst 33342 (0.5 μg/mL) for 1 h and washing with PBS, the insert was removed and the migration of cells into the cell-free zone was screened. Time-lapse images were captured in 20 min intervals for 8 h at 37°C and 5% CO2 using the PerkinElmer Operetta (PerkinElmer, Inc., Waltham, MA, United States) high-content imaging system with a 20 × objective (20 × long WD; NA = 0.45, working distance: 7.8 mm; field of view: 675 × 509; depth of focus: 4.6 μm; optical xy resolution: 0.7 μm).
5
High Content Imaging of Transfected Cells
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Microplates with seeded and transfected cells (Corning 96-well plate) were imaged on the High Content Screening System Operetta™ (PerkinElmer). In each well, images were acquired in 9 preselected fields with LWD 10x objective over four channels: brightfield, digital phase contrast (DPC) based on brightfield images, with excitation filters 460–490 and 520–550 nm and emission filters 500–550 and 560–630 nm for GFP and mCherry reporters, respectively. For the feature extraction, the images were analysed by Harmony software version 4.1 (PerkinElmer). Briefly, individual cell nuclei were segmented in the DPC channel. Nucleus morphology, GFP, and mCherry mean intensity were quantified in the cell nuclei population. Single-cell object features were extracted from each sample well. To discriminate between GFP/mCherry negative and positive cells, a threshold was determined based on the GFP/mCherry intensity frequency distribution of all samples for each experiment.
6
Quantifying Cell Infection Ratio
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The images acquired with Operetta (Perkin Elmer) were analyzed using our in-house Image-Mining (IM) software to quantify cell numbers and infection ratio by counting Hoechst-stained nuclei and viral N protein-expressing cells, respectively. The infection ratio of each well was normalized to the average of infection percentage of infection group (0.5% DMSO) and mock infection group in each plate. The cell ratio was determined according to the number of cells of each well versus the average number of cells of mock infection in each plate and described as “cell number to mock” in the dose–response curve (DRC) graph. The DRCs of plitidepsin (both. dissolved either in DMSO or in ampoule), was analyzed using the XLfit equation: Y = Bottom + (Top − Bottom)/(1 + (IC50/X)Hillslope).
The IC50 and 50% toxicity concentration (CCso) values were calculated from the fitted DRCs. Selectivity index (SI) was calculated as CC50/IC50. All IC50 and CC50 values were determined in duplicate experiments. All the experiments were conducted simultaneously.
The IC50 and 50% toxicity concentration (CCso) values were calculated from the fitted DRCs. Selectivity index (SI) was calculated as CC50/IC50. All IC50 and CC50 values were determined in duplicate experiments. All the experiments were conducted simultaneously.
7
Quantifying Adipocyte Lipid Droplets
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Adipocytes cultured in 96-well opaque black plates were fixed with 4% PFA for 20 min, washed three times with PBS and stained with Bodipy (Invitrogen) for LDs and Hoechst 33432 (Cell Signaling) for nuclei. Images were captured via an automated system (Operetta; PerkinElmer) and analysed using the Harmony software by calculating the ratio of cells with lipid droplet to the total cell number as described before39 (link).
8
High-throughput Antiviral Screening of FDA-Approved Drugs
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US FDA-approved drug library which contains 1,700 compounds were purchased from TargetMol (Massachusetts, USA). LLC-MK2 cells were seeded at 2 × 104 cells per well in 96-well plates and incubated at 37°C and 5% CO2. The next day, LLC-MK2 cells were treated with the compounds at a concentration of 10 μM. After 1 h of treatment, cells were infected with OC43 at MOI of 1. At 48 h post-infection (hpi), cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Immunofluorescence staining was performed using mouse anti-OC43 NP antibody, followed by anti-mouse Alexa Fluor 488 and DAPI (Sigma, St. Louis, MO). Images were captured by Operetta (PerkinElmer, Massachusetts, USA) at the magnification of 20× objective. The infection ratios were calculated using automated image analysis software (Harmony 3.5.2, PerkinElmer). Remdesivir and DMSO were used as positive and negative controls, respectively.
The positively identified drugs from this screen were used to perform dose-response curves against OC43 on LLC-MK2 and against SARS-CoV-2 using Vero cells as described below.
The positively identified drugs from this screen were used to perform dose-response curves against OC43 on LLC-MK2 and against SARS-CoV-2 using Vero cells as described below.
9
High-Content Imaging for LiPD/DiPD Control
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For LiPD background depletion control, GFP-CXXC4 cells stably expressing LiPD were mixed with cells lacking the LiPD and seeded on coverslips. For testing of DiPD background control, GFP-PCNA cells with and without stably expressed DiPD were mixed and seeded on coverslips. Cells were fixed and HA was stained, microscopic slides were prepared as described before.
For high-content analysis, samples were imaged with an automatic fluorescence microscope (Operetta, Perkin Elmer). DAPI, GFP and Alexa Fluor 594 (anti-HA) were excited with correspondent lasers using a ×40 high NA objective. For each coverslip 121 fields were imaged (an 11 × 11 fields square area). Image analysis was performed with the Harmony analysis software (Perkin Elmer). In brief, cell nuclei were recognized and segmented in the DAPI channel and correspondent GFP and anti-HA fluorescence intensities were measured for each cell nucleus. The cells then were divided into two groups (LiPD or DiPD group versus control group) according to the anti-HA fluorescence intensity. The GFP intensity of each group was plotted in boxplot format with R (Rstudio).
For high-content analysis, samples were imaged with an automatic fluorescence microscope (Operetta, Perkin Elmer). DAPI, GFP and Alexa Fluor 594 (anti-HA) were excited with correspondent lasers using a ×40 high NA objective. For each coverslip 121 fields were imaged (an 11 × 11 fields square area). Image analysis was performed with the Harmony analysis software (Perkin Elmer). In brief, cell nuclei were recognized and segmented in the DAPI channel and correspondent GFP and anti-HA fluorescence intensities were measured for each cell nucleus. The cells then were divided into two groups (LiPD or DiPD group versus control group) according to the anti-HA fluorescence intensity. The GFP intensity of each group was plotted in boxplot format with R (Rstudio).
10
Immunocytochemical Quantification of p53 and Oct4
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Cells were fixed in 2% paraformaldehyde and stained with 10 μg/mL Hoechst 33342 (Invitrogen) with a Combi Multidrop Dispenser (Thermo). Standard fluorescence immune-cytochemical techniques were used to stain the cells with a monoclonal antibodies for p53 (Cell Signaling Technologies) and for Oct4 (BD), and Alexa-Fluor-488 and Alexa-Fluor-546 secondary antibodies (Invitrogen). Immuno-cytochemical staining was performed by a Janus automated liquid handler (Perkin Elmer). Images were acquired at 20x with an Operetta (Perkin Elmer) using standard filter sets. Image analysis was performed using custom scripts in Acapella software (Perkin Elmer). Nuclear objects were segmented from the Hoechst signal. The fraction of nuclear-localised Alexa-Fluor-488- and Alexa-Fluor-546-positive cells was quantified. Images and well-level data were stored and analysed in a Columbus Database (Perkin Elmer).
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