Microencapsulated hPSC-Hep spheroids were washed in PBS and briefly stored in CMRL-1066 serum-free medium prior to implantation. Implantation of alginate encapsulated cells and empty alginate beads was performed as previously described (King et al., 2001 (link); Bochenek et al., 2018 (link)). The intraperitoneal space was selected for the implantation site due to proximity to the liver and easy retrieval of the microbeads by saline lavage upon completion of the experiments. The implantation of microencapsulated hPSC-Hep spheroids was performed on 16 immunocompetent C57BL/6J male mice aged 16 weeks; empty alginate microbeads were transplanted into 8 mice as a control. The microbeads were retrieved from the peritoneal cavity after 10 (N = 12) and 20 (N = 12) days. The intervention (sulfated alginate) and control (unmodified alginate), and no-cell control groups were matched in size (n = 4 each). Images of the microbeads were collected using brightfield imaging by Operetta CLSTM High-Content Analysis System (Perkin Elmer), and the extent of PFO was evaluated manually in ImageJ.
Operetta clstm high content analysis system
Manufactured by PerkinElmer
Sourced in United States
The Operetta CLS High-Content Analysis System is a fully automated, high-throughput imaging and analysis platform designed for cell-based assays. It combines high-resolution imaging with powerful image analysis capabilities to enable quantitative assessment of cellular phenotypes and functions.
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6 protocols using operetta clstm high content analysis system
1
Implantation of Microencapsulated hPSC-Hep Spheroids
2
Alginate Microbeads Permeability Assay
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The permeability of sulfated alginate was characterized by the extent of diffusion of FITC probes conjugated to dextran samples with defined molecular sizes. Samples of alginate microbeads were treated with 50 μg/ml solution of FITC-dextran in a range of molecular sizes ranging from 4 kDa to 2 MDa. The samples were incubated for 1 h, 15 and 24 h, and were imaged by Operetta CLSTM High-Content Analysis System (Perkin Elmer) using ×6 magnification and 488 nm excitation laser. The z-height of the plane was selected manually to obtain images penetrating the center of the microbeads. Permeability was measured semi-quantitively in ImageJ (Schneider et al., 2012 (link)) by comparing the mean pixel intensity values from the defined central point of each microbead to the mean pixel intensity values of the background (microbead-free area) (n ≥ 11 per group).
3
MCMV Infection Immunofluorescence Assay
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MCMV-infected M2-10B4 cells in 96-well plates were fixed with 4% paraformaldehyde and washed with PBS followed by permeabilization with 0.2% Triton X-100/PBS, and then incubated with a blocking solution of 5% goat serum/0.3% Triton X-100/PBS for 1 h. Plates were probed overnight with an antibody against MCMV m123/IE1 (CROMA101; 1:1000; University of Rijeka). Antibody was visualized using goat-anti-mouse Alexa Fluor 488 secondary antibody (1:1000; Thermo Fisher Scientific) and nuclei were stained with DAPI (BD). Images were acquired using the Operetta CLSTM high-content analysis system (Perkin Elmer) with a ×10 objective in confocal mode. At least 20 out of 25 fields per well were captured and analyzed for DAPI and/or Alexa Fluor 488-positive cells by Harmony software version 4.9. Representative images were captured with a ×20 objective in confocal mode.
4
Lung Immune Cell Quantification
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Tissue slides were deparaffinized and dehydrated as explained above. Specific lymphocyte and monocyte/macrophage lung infiltration were evaluated by immunofluorescence measuring CD3 (100244 Biotin anti-mouse CD3) and CD80 (104748 Ultra-LEAFTMPurified anti-mouse CD80) surface markers. Fluorescent detection of the primary antibodies was performed using goat anti-Armenian hamster IgG H&L (Abcam AB173004, Alexa Fluor® 647) and tomato red tagged streptavidin protein. DAPI was used for nuclear staining and anti-podoplanin monoclonal antibody (AF488-conjugated) was used for membrane surface. The lung tissues were then imaged using the Operetta® CLSTM High Content Analysis System (PerkinElmer, Waltham, MA, USA). Specific parameters for tissue detection and identification were established. Data analysis was performed using the Harmony 4.8 software from PerkinElmer.
5
High-Content Analysis of ARV-Induced Syncytia
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The cells were screened and analyzed with the Operetta CLSTM High-Content Analysis System (PerkinElmer, MA, United States). All the images were automatically analyzed using Harmony® software with PhenoLOGIC (PerkinElmer, MA, United States) following the manufacturer’s instructions. To measure cell membrane cholesterol levels, images were acquired from 49 fields per well in the DAPI (filipin III shares a wavelength range with DAPI), brightfield, and digital phase control (DPC) channels 40 × objective. The fluorescence intensity of filipin III in the cell membrane was used to determine the cell membrane cholesterol content. For ARV μNS marker and syncytium analysis, images were collected from 25 fields per well in the FITC, DAPI, brightfield, and DPC channels with a 20× objective. Based on the histograms of the background fluorescence intensity from the mock-treated group, the populations presented with FITC intensities higher than 900 were identified as ARV-positive cells. For syncytium analysis, the cell nuclei were stained with DAPI, and multinuclear cells enclosed within a single plasma membrane (number of nuclei ≥ 3) were considered syncytia.
6
High-Content Imaging with Operetta CLS
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Image acquisition was conducted on an Operetta CLS TM high-content analysis system (PerkinElmer, Waltham, MA) with binning 1, using a 40× water immersion objective in wide-field mode with Z-stack of 4 planes and distance steps of 1.0 µm. Four to 16 fields of view and six channels were captured.
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