GFP-expressing PDE10 stable knockdown and vector control cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cell nuclei were counterstained with 4′, 6-diamidino-2-phenyl-indole (DAPI; 1 mg/ml). Images were captured by the Operetta confocal microscope (Perkin Elmer, Waltham, MA, USA).
Operetta confocal microscope
Manufactured by PerkinElmer
Sourced in United States
The Operetta confocal microscope is a high-content imaging system designed for advanced cell analysis. It provides high-resolution, confocal imaging capabilities to capture detailed cellular information. The system is equipped with multiple excitation lasers and detectors to enable imaging of a variety of fluorescent markers.
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3 protocols using operetta confocal microscope
1
Visualizing PDE10 Knockdown in Cells
2
Visualizing PDE10 Knockdown in Cells
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GFP-expressing PDE10 stable knockdown and vector control cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cell nuclei were counterstained with 4′, 6-diamidino-2-phenyl-indole (DAPI; 1 mg/ml). Images were captured by the Operetta confocal microscope (Perkin Elmer, Waltham, MA, USA).
3
Phenotypic Validation of Immune Cell Migration
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To confirm the phenotypic identity of the cells following treatment, 1 × 105 MDMs were loaded on top of the aforementioned Transwell Permeable Inserts (BD Biosciences, Franklin Lakes, NJ #353493, USA), which contained a pre-grown and differentiated, pseudostratified epithelial layer at ALI. They were then transferred into wells that contained media plus different concentrations of dexamethasone, and 50 ng/mL rhGM-CSF was applied as a positive chemoattractant to the lower chamber. Incubation was then carried out for 24 h at 37 °C, after which the membranes were cut out, stained, fixed, and mounted on slides (ThermoFisher Scientific, Waltham, MA #WT4301672, USA) and coverslips (VWR, Radnor, PA #631-0657) with Mowiol (Carl Roth, Karlsruhe, Germany #0713.2) applied as the essential mounting medium. Immune cell migration characteristics were analyzed using computational 3D imaging (Perkin Elmer, Operetta Confocal Microscope, Waltham, MA, USA). The protocol was a loose adaption from [21 (link)].
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