Imaging experiments were performed using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany), and analysed with Harmony 4.6 software (PerkinElmer). To assess the tubulin integrity, we acquired HeLa cells images using 63× magnification, taking 25 fields per sample in biological and technical triplicates. Data were analysed using the following building blocks: 1—Find Nuclei, 2—Find Cytoplasm (Tubulin+). IC50 was obtained by counting the number of nuclei in each field. Cell dimensions were assessed using the following building blocks: 1—Find Nuclei, 2—Find Cytoplasm (Tubulin+), 3—Calculate Morphology properties (Area), as previously described.37–39 (link) IC50s were calculated using the number of cells detected in every well as indexing parameter by Harmony 4.6 software as described here.40 (link)
Operetta cls high content imaging device
Manufactured by PerkinElmer
Sourced in Germany
The Operetta CLS high-content imaging device is a laboratory instrument designed for automated, high-throughput imaging and analysis of cell samples. It is capable of capturing and processing images of cells and subcellular structures at high resolution.
Automatically generated - may contain errors
Lab products found in correlation
Harmony 4, by PerkinElmer (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Inverted microscope, by Zeiss (2 mentions)
Anti sars cov 2 spike protein, by Sino Biological (1 mentions)
Cellcarrier ultra plates, by PerkinElmer (1 mentions)
Oil red o, by Merck Group (1 mentions)
Air objective, by Zeiss (1 mentions)
Observer z1, by Zeiss (1 mentions)
6 protocols using operetta cls high content imaging device
1
Evaluating Tubulin Integrity and Cell Morphology
2
SARS-CoV-2 Infection and Lipid Droplet Analysis
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Imaging experiments were performed using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany) and were analyzed with Harmony 4.6 software (PerkinElmer). Huh-7 cells were seeded in 96-well CellCarrierUltra plates (Perkin Elmer, Hamburg, Germany) 24 h prior to treatment or infection, as described above. Cells were fixed in ice-cold methanol and stained 48 h after infection with the following primary antibodies: Anti-SARS-CoV-2 Spike protein (#40588 RC02, Sino Biological, Beijing, China, 1:200). Lipid droplets (LDs) were stained using Oil Red O (1:5000 diluted in water) for 15 min (Sigma-Aldrich, St. Louis, MO, USA). Nuclei were stained with DAPI (1 µg/mL). To quantify the number of infected cells, the following building block was used: find nuclei, find cytoplasm, calculate intensity properties (SARSCoV2-S1-Alexa 488), select population of infected cells, and find SARS-CoV-2-positive (Alexa 488+) cells. Around 140 fields per well per experiment were analyzed using a 63× water objective. To quantify the number of LDs, we used a detailed protocol described previously [32 (link),33 (link)].
3
High-Content Imaging of 3D Spheroids
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Microscopy analysis of spheroids was done using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany) equipped with laser-based autofocus. Spheroids were imaged using Z-stacks to generate maximum intensity projections using Harmony 4.9 quantitative imaging analysis software (PerkinElmer, Hamburg, Germany). The software was also used to segment regions of the spheroid, such as the core and outgrowth area, using image algorithms only. Fluorescence of sytox green (final concentration of 1 µM incubated for 15 min before imaging; ThermoFisher, Dreieich, Germany) was captured using appropriate excitation and emission wavelengths. For live-cell imaging over 24 h, the device was heated to 37 °C, and 5% CO2 was supplied. Evaporation protection was achieved by filling the outer wells of the 96-well plate with double-distilled water.
4
High-Content Imaging of Cellular Samples
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Selected conditions were imaged using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany). Brightfield and digital phase contrast were imaged using a 5x air objective (NA: 0.16; Zeiss, Jena, Germany) and overlaid for display.
5
Micro-insert Migration Assay for PDA Cells
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The 2-well micro-insert (Ibidi, Gräfelfing, Germany) was added to the center of a well of 24-well plates. To each chamber, 1 × 104 PDA cells were added and allowed to adhere overnight. Then, each side was plasma-treated, and the chamber was lifted to allow the analysis of cells migrating into the 400–500 µm-wide gap. Brightfield imaging was performed at 0 and 24 h using an inverted microscope at 200× magnification (Carl Zeiss, Jena, Germany). Cell migration within these 24 h was reconstructed using overlay images. The total growth area of untreated and plasma-treated PDA cells was analyzed using Harmony 4.9 imaging software (PerkinElmer, Hamburg, Germany). The cellular area was determined using digital phase-contrast imaging using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany). This device and software were also used to determine mean speed during kinetic live-cell imaging of PDA cells, for which 2.5 × 103 PDA cells were seeded, plasma-treated, and incubated for 1 h before the addition of 2.5 × 103 RAW cells. For imaging of cluster formation induced by PDA-induced M2 macrophage polarization [99 (link)], 2 × 105 PDA and RAW cells were seeded in wells of 6-well plates and imaged at 72 h using an Observer Z.1 (Carl Zeiss, Jena, Germany). Analysis of cluster numbers was performed using ImageJ software (Wayne Rusband, NIH; open source).
6
Quantitative High-Content Imaging of Mitochondrial Morphology
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Imaging experiments were performed using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany), and analysed with Harmony 4.6 software (PerkinElmer). To investigate LC3-II red spots, we analysed cells using 63x magnification, taking 25 fields per sample in biological and technical triplicates. Data were analysed using the following building blocks: 1- Find Nuclei, 2- Find Cytoplasm of transfected cells (RFP- and GFP-positive) 3- Find RFP+ spots. Mitochondria morphology was first assessed analysing the mitochondria intensity of MitoSOX using the following building blocks: 1—Find Nuclei, 2—Find Cytoplasm (MitoSOX+) 3—Find MitoSOX+ spots (mitochondria) 4—Calculate Intensity Properties on “mitochondria” population. Then, SER analysis was performed to evaluate the alteration in mitochondria morphology. Briefly, cells were segmented using the SER algorithm for the following parameters: hole, edge, ridge, valley, saddle, roundness and length. SER analysis allows texture-based read out of pixel distributions using fluorescence based or brightfield images. For all types of quantitative image analysis, 27 fields of views (FoV) were acquired for each replicates per condition.
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