The compound library was formatted into groups of 96-well plates based on excitation/emission spectra (Table 1). Screening was performed in batches of 12–15 plates, with each compound group screened in the same batch. The compounds (prepared in DMSO as 100 μM stock solutions) were added to the medium of 14–16 DIV cortical neurons via automated liquid handling system (Cell Explorer; PerkinElmer) for a final concentration of 2 μM. Following 20 min incubation at 37 °C, plates were washed 3× at room temperature in normal Tyrodes solution (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, 25 mM Hepes pH 7.4), and images acquired at room temperature in four fields of view/well using the high-throughput screening microscope (IN Cell Analyzer; GE Healthcare, 20× objective) and CCD camera (2048 × 2048 pixels) in the Columbia Genome Center HTS facility. Following screening, plates were fixed with 4% formaldehyde in PBS, washed once in PBS, and stored in the dark at 4 °C.
Cell explorer
Manufactured by PerkinElmer
The Cell::Explorer is a high-content cellular imaging system designed for live-cell analysis. It provides automated image acquisition, analysis, and data management capabilities to support a wide range of cell-based assays and cellular research applications.
Automatically generated - may contain errors
Lab products found in correlation
Variomag, by Thermo Fisher Scientific (2 mentions)
Multidrop combi liquid dispenser, by Thermo Fisher Scientific (2 mentions)
In cell analyzer, by GE Healthcare (1 mentions)
Echo 520, by Beckman Coulter (1 mentions)
Nanohead, by PerkinElmer (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
384 pin tool, by V&P Scientific (1 mentions)
Janus automated workstation, by PerkinElmer (1 mentions)
4 protocols using cell explorer
1
High-throughput Compound Screening in Neurons
2
Dose-Response Validation of Luciferase Assay
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The dose-response validation was done in an extended set of cell-based luciferase reporter assays. Fresh compound solutions of those active in the primary screens were retrieved from the frozen library and diluted in DMSO to ECHO-qualified 384-well cyclic olefin copolymer plates (Labcyte). On the day of the experiment, reporter cells were harvested and resuspended into phenol red-free DMEM culture media at a concentration of 500 cells/μl. Four-microliters aliquots of the cell suspension were dispensed to white polystyrene 1536-well plates (Corning) by a Multidrop Combi liquid dispenser (Thermo Scientific). The test compounds were transferred from the 384-well compound plates to 1536-well assay plates using contact-free acoustic transfer by ECHO 520 (Labcyte) integrated in the fully automated robotic HTS station Cell::Explorer (Perkin Elmer). The compounds were tested in triplicates at eight different concentrations in the range from 10 μM to 5 nM. Each plate was shaken for 30 s using the plate shaker Variomag (Thermo Scientific) and then incubated at 37°C and 5% CO2 in humidified atmosphere for 24 h.
3
High-throughput drug screening in cells
4
High-Throughput Cell-Based Screening Protocol
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The primary screening was carried out in a fully automated robotic platform Cell::Explorer (Perkin Elmer) in a 384-well format. On the day of the experiment, reporter cells were harvested and resuspended into phenol red-free DMEM culture media to obtain a concentration of 200 cells/μl. Twenty-five microliters aliquots of the cell suspension were dispensed to white polystyrene 384-well plates (Corning) by a Multidrop Combi liquid dispenser (Thermo Scientific). The test compounds were transferred from the 384-well compound plates to 384-well assay plates in Janus Automated Workstation (Perkin Elmer) integrated to Cell::Explorer and equipped with 384 Pin tool (V&P Scientific). The final concentration of the screened compounds was 1 μM. Each plate was shaken for 30 s using a plate shaker Variomag (Thermo Scientific) and then incubated at 37°C and 5% CO2 in humidified atmosphere for 48 h.
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