Drug screening was carried out as previously described (Communications Biology, in press) Tac12 infected macrophages expressing a GFP-CLASP fusion protein15 (link) were plated in 96-well plates and treated with the compounds at 10 μM for 48 h. Immunofluorescence was performed on fixed cells using a specific anti-H3K18me1 antibody to label parasite nuclei. Cells were incubated with DAPI to detect host and parasite nuclear DNA. The parasite surface membrane was monitored by GFP-CLASP fluorescence15 (link). Image capture (30 fields per condition) and analysis was performed with the Opera Phenix microscope (Perkin Elmer, Photonic BioImaging platform, Pasteur Institute) and the associated Acapella Software to monitor host and parasite survival.
Opera phenix microscope
Manufactured by PerkinElmer
Sourced in United Kingdom
The Opera Phenix microscope is a high-content screening system designed for advanced cell imaging and analysis. It is capable of capturing high-resolution images of cells and cellular structures across multiple wavelengths and timepoints. The Opera Phenix provides researchers with a versatile platform for a wide range of applications, including drug discovery, cell biology, and phenotypic screening.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst h33342, by Thermo Fisher Scientific (3 mentions)
Harmony 4.9 high content imaging and analysis software, by PerkinElmer (3 mentions)
Water immersion lens, by PerkinElmer (2 mentions)
Ec plan neofluar x10 air objective na 0.3 wd 5 2mm, by Zeiss (2 mentions)
W plan apochromat x20 water objective na 1.0 wd 1 17mm, by Zeiss (2 mentions)
Sensoplate, by Greiner (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cellcarrier, by PerkinElmer (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Ag 25b 0006, by Adipogen (1 mentions)
Harmony high content imaging analysis software, by PerkinElmer (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Image it tmrm reagent, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Laminin, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Anti cenpf, by Abcam (1 mentions)
19 protocols using opera phenix microscope
1
Screening for Anti-Parasite Compounds
2
High-throughput Imaging of DNA Repair Foci
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For high-throughput imaging and analyses of focus formation by RPA2, RAD51 and PALB2 proteins, cells were plated on 96-well Cell Carrier (Perkin Elmer) plates at a density of 7500−10,000 cells/well. The following day, cells were either mock-treated or irradiated (6 Gy) and processed for imaging as described under Immunofluorescence method. Imaging was performed on the spinning disk Opera Phenix microscope (Perkin Elmer) using either ×20 or ×40 water immersion objectives. Images were acquired in single optimised focal plane for each of the three channels (405, 488 and 568 nm) in the confocal mode. Image analysis and evaluation was done using Harmony High-Content Imaging and Analysis software. In each experiment, 7−10 fields (>500 cells) per well were acquired and analysed.
3
High-Content Live-Cell Imaging Protocol
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High content live-cell imaging: 30,000 NRK cells were seeded into a 96-well olefin-bottom imaging plate (Perkin Elmer, Cat# 6055302). The plate was placed in a pre-heated (37 °C) Opera Phenix microscope with a 60× water-immersion lens (Perkin Elmer) at 5% CO2. Images were acquired at 1080 × 1080 pixels using Harmony software.
4
Time-lapse Imaging of 3D Co-cultures
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CBF-0.5 hydrogels containing mPCOs, PaFs and BMDMs 1:5:5 ratio of PDO:Paf:BMDM were prepared as described previously and crafted into 10 μL domes in 24-well Sensoplate (Greiner). Co-culture domes were imaged via the PerkinElmer Opera Phenix microscope. A Zeiss EC Plan Neofluar X10 air objective NA 0.3 WD 5.2mm or Zeiss W Plan-Apochromat X20 water objective NA 1.0 WD 1.17mm were used as indicated. Pre-scan directed z-stack acquisition of individual spheres within the gel was used and co-cultures were imaged every hour over a time-window of 72h starting from day three of culture. Videos and representative still images were generated from maximum intensity projections using Harmony 4.8 software. Representative images were exported from Harmony 4.8 in the portable network graphic format with scaling equally applied to all conditions.
5
Microglia ASC Specks and Astrocyte NFκB
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The number of ASC specks within microglia and the subcellular localisation of NFκB within astrocytes were assessed by immunocytochemistry in cells seeded in 96-well plates. After treatment, cells were washed with DPBS (Gibco) and fixed in 4 % (v/v) PFA for 20 min at RT prior to further washing. Cells were permeabilised and non-specific binding blocked using 0.1 % (v/v) Triton-X-100, 5 % (w/v) BSA in TBS for 1 h at ambient temperature before incubating with an anti-ASC antibody (1:200, Adipogen, AG-25B-0006) or anti-NFκB (p65 subunit, 1:400, Cell signalling, D14E12) overnight at 4 °C. AlexaFluor 488 conjugated secondary antibody (Invitrogen) was added for 1 h at RT. Nuclei were stained with Hoechst-33342. Cells were imaged using a confocal Opera Phenix microscope (Perkin Elmer). The number of ASC specks per well was normalised to the number of nuclei and the mean nuclear intensity of NFκB was normalised to the total cell intensity per well using Harmony High-Content imaging analysis software (Perkin Elmer).
6
High-Content Imaging of Mycobacterial Infection
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30,000 iPSDM were seeded into a ViewPlate glass bottom 96- well plate and treated with LLOMe, Silica or infected with Mycobacterium tuberculosis as described above. The plate was sealed with parafilm and placed in a pre-heated (37 °C) Opera Phenix microscope with 40× or 60× water-immersion lens (PerkinElmer) with 5% CO2.
Capture settings were: Image-iT TMRM Reagent (I34361, Thermo Fischer) and MitoTracker Red CMXRos (M7512, Thermo Fischer) were excited with the 561 nm laser at 10% power with 100 ms exposure. MitoTracker Deep Red FM (M22426, Thermo Fischer), iABP probe and Mtb E2crimson were excited with the 640 nm laser at 10% power with 100 ms exposure. The mitoTimer construct was excited with the 488 nm laser and the 561 nm laser at 10% power and 100 ms exposure. The pHyPer-dMito construct was excited with the 405 and 488 nm lasers and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1020 × 1020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Cystatin B c-GFPSpark-tag, RAB7-GFP, RAB7(Q67L)-GFP and Lamp1-mNeonGreen- expressing cells were excited using the 488 nm laser at 10% power with 50 ms exposure. Hoechst H33342 (H3570, Thermo Fischer) was excited using the 405 nm laser at 15% power with 100 ms exposure.
Capture settings were: Image-iT TMRM Reagent (I34361, Thermo Fischer) and MitoTracker Red CMXRos (M7512, Thermo Fischer) were excited with the 561 nm laser at 10% power with 100 ms exposure. MitoTracker Deep Red FM (M22426, Thermo Fischer), iABP probe and Mtb E2crimson were excited with the 640 nm laser at 10% power with 100 ms exposure. The mitoTimer construct was excited with the 488 nm laser and the 561 nm laser at 10% power and 100 ms exposure. The pHyPer-dMito construct was excited with the 405 and 488 nm lasers and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1020 × 1020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Cystatin B c-GFPSpark-tag, RAB7-GFP, RAB7(Q67L)-GFP and Lamp1-mNeonGreen- expressing cells were excited using the 488 nm laser at 10% power with 50 ms exposure. Hoechst H33342 (H3570, Thermo Fischer) was excited using the 405 nm laser at 15% power with 100 ms exposure.
7
Assessing Stromal Cell-Mediated Cytotoxicity
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This section describes the experiment shown in Figures 1 D, 1E, S1 , S5 , and S9 . We labeled leukemia cells from four or six other patients with CellTracker Green and cocultured them (2 × 105 cells/well) with CellTracker Blue labeled NKTert (1 × 104 cells/well) or HS-5 (2 × 104 cells/well) stromal cells. CLL cells were pretreated with solvent control or 63 nM venetoclax or 63 nM venetoclax and 10 μM Z-VAD-FMK (Sigma-Aldrich) for 24 hours. For chloroquine treatment, NKTert cells were pretreated with 20 μM hydroxychloroquine for 24 hours. Co-culturing was performed for 16 hours. Cultures were additionally stained with lysosomal dye NIR and PI before imaging. The samples were imaged on an Opera Phenix microscope (Perkin Elmer) in confocal mode. For clearer visualization, the signal from the lysosomal dye is not shown in Figure 1 D, while CellTracker Green signal and PI staining are not shown Figure 1 E. The images shown are representative of all experiments.
8
Analyzing Neuronal Morphology and Dendritic Spines
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The neuronal morphology of 14 DIV neurons previously transfected with the plasmid peGFP-N1 (Clontech) on 5–7 DIV and treated on 13 DIV was assessed using high resolution digital images of live neurons taken using an Opera-Phenix microscope (PerkinElmer). Neurite complexity was analysed using Harmony software: total and maximum neurite length, and number of nodes and extremities were quantified and compared among groups. NeuronStudio software (CNIC, Mount Sinai School of Medicine) was used for dendritic spine analysis. Spine density was defined as number of spines per micrometer of dendrite length. Dendritic spine densities were calculated from 20 neurons/condition.
9
Automated High-Content Imaging of MCF7 Cells
10
Monitoring Apoptosis in Leukemia Cells
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This section describes the experiment shown in Figure 2 C. Primary CLL cells were labeled with CellTracker Green. Apoptosis was induced by treatment with 63 nM venetoclax. After 24 hours the cells (2 × 105 cells/well) were added to primary MSCs of four different healthy donors (1 × 103 cells/well), labeled with CellTracker Blue into 96-well glass bottom microscopy plates (zell-kontakt GmbH). After incubation for 16 hours the cultures were stained with lysosomal dye NIR and PI. The samples were imaged on an Opera Phenix microscope (Perkin Elmer) in confocal mode. Representative images are shown.
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