Cells were fixed by 4% paraformaldehyde (Sigma Aldrich) for 15 min, permeabilized by 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, blocked by 3% bovine serum albumin (BSA, Sigma-Aldrich) for 20 min, and stained by anti-TUBB3 (neuronal class III β-tubulin) (1:1000; Covance, Princeton, NJ, USA) antibody at 4° C overnight. The cells were washed by phosphate-buffered saline (PBS) for twice and stained with the secondary Alexa Fluor ®555 goat anti-rabbit antibody (1:1000; Molecular probes) at room temperature for 3 h, and with 4’-6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml, Sigma-Aldrich) for 30 min. Images of neurites were captured by Micro Confocal High-Content Imaging System (Molecular Devices), and analyzed by MetaXpress Neurite Ougrowth Application Module (Molecular Devices).
Micro confocal high content imaging system
Manufactured by Molecular Devices
Sourced in United States
The Micro Confocal High-Content Imaging System is a laboratory equipment designed for imaging and analysis of cellular samples. It utilizes confocal microscopy technology to capture high-resolution, three-dimensional images of biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (2 mentions)
Alexa fluor 555 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti tubb3, by Fortrea (1 mentions)
Tubulin, by Fortrea (1 mentions)
Lsm 880 with airyscan, by Zeiss (1 mentions)
Sh sy5y, by Merck Group (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Metaxpress image acquisition and analysis software, by Molecular Devices (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cellrox reagent, by Thermo Fisher Scientific (1 mentions)
Imagexpress, by Molecular Devices (1 mentions)
Curcumin, by Merck Group (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
4 protocols using micro confocal high content imaging system
1
Immunofluorescence Staining of Neurites
2
Live-Cell Imaging in Controlled Environment
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Live-cell imaging was carried out in a temperature (37°C) and CO2-controlled environment using a Micro Confocal high-content imaging system (Molecular Devices, USA) or laser-scanning confocal LSM 880 with airy scan (Zeiss, Germany).
3
Neuroprotective Flavones Screening Assay
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Aβ-GFP SH-SY5Y (2.5×104 cells/well) on 96-well plates were given all-trans-retinoic acid (10 µM; Sigma-Aldrich) to induce neuronal development. On a subsequent day, the cells were pre-treated with test flavones (1-5 µM) for 8 h, and doxycycline was added to induce Aβ-GFP expression. Treatments including retinoic acid, the test flavones and doxycycline were refreshed every two days. On day 8, after nuclear staining (0.1 g/mL Hoechst 33342; Sigma-Aldrich), images of the stained cells at 482 nm excitation and 536 nm emission wavelengths were taken and inspected using Micro Confocal High-Content Imaging System and MetaXpress Image Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA, USA). EC50 values of test compounds were calculated as described.
In addition, cells were exposed at 37°C for 30 min with cell-permeant CellROX Deep Red reagent (5 μM; Molecular Probes, Eugene, OR, USA) and reactive oxygen species (ROS) were assessed using the high content analysis system at excitation/emission wavelengths of 644/665 nm. EC50 values of test compounds were calculated as described.
In addition, cells were exposed at 37°C for 30 min with cell-permeant CellROX Deep Red reagent (5 μM; Molecular Probes, Eugene, OR, USA) and reactive oxygen species (ROS) were assessed using the high content analysis system at excitation/emission wavelengths of 644/665 nm. EC50 values of test compounds were calculated as described.
4
Curcumin and SG-Tang Modulate Aβ-GFP Aggregation
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Aβ-GFP SH-SY5Y cells (2.5 × 104) were seeded into a 96-well plate with retinoic acid (10 μM; Sigma-Aldrich) on day 1 [74 (link)]. On the next day, curcumin (1.2–5 μM; Sigma-Aldrich), a potent inhibitor against Aβ aggregations as a positive control [75 (link)], or SG-Tang (1–100 μg/ml) were added for 8 h. Doxycycline (5 μg/ml) were added to induce Aβ-GFP expression for another 7 days. Then cells stained with Hoechst 33342 (0.1 μg/ml, Sigma-Aldrich) were captured by Micro Confocal High-Content Imaging System (Molecular Devices, Synnyvale, CA, USA) at excitation/emission wavelengths of 482/436 nm, and analyzed by ImageXpress (Molecular Devices). To measure ROS, cells were incubated with the reddish fluorogenic CellROX reagent (5 μM; Molecular Probes) and Hoechst 33342 at 37° C for 30 min. Micro Confocal High-Content Imaging System at excitation/emission wavelengths of 640/665 nm and ImageXpress were used for the acquisition and analysis of cell images.
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