After induction of adipogenic differentiation for 2 days and following AAP treatment for 7 days, cells were fixed in phosphate-buffered formaldehyde (4%; pH 7) for 15 min and stained with a BODIPY 493/503 fluorescence lipid probe for 15 min as previously described53 (link). The nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI; Invitrogen, Thermo Fisher Scientific Inc.). Images of lipid droplets were captured using an inverted microscope (Olympus Corporation, Tokyo, Japan) or the ImageXpress® Micro XLS Widefield High-Content Analysis System (Molecular Devices, Sunnyvale, CA, USA). The percentage of lipid droplets in each group was normalized to total number of cells determined by the DAPI counterstain. Data analysis was performed using the MetaXpress software package (Molecular Devices).
Metaxpress software package
Manufactured by Molecular Devices
MetaXpress is a comprehensive software package designed for image acquisition, analysis, and management in high-content screening (HCS) applications. The software provides a suite of tools for automated image capture, multi-parametric analysis, and data visualization to support cellular and subcellular-level measurements in a wide range of experimental settings.
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Lab products found in correlation
Imagexpress micro xls widefield high content analysis system, by Molecular Devices (2 mentions)
Imagexpress micro confocal cellular imaging system, by Molecular Devices (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Inverted microscope, by Olympus (1 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fv10i laser confocal microscope, by Olympus (1 mentions)
Image it fix perm kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using metaxpress software package
1
Quantifying Lipid Droplet Formation
2
Quantitative High-Content Imaging Cytotoxicity
3
Intracellular SREBP-1 Quantification Protocol
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Intracellular SREBP-1 staining was performed using Image-iT® Fix Perm Kit (Thermo Fisher Scientific Inc.). Briefly, cells were fixed and permeabilized in Fixation Buffer and Permeabilization Buffer for 15 min, respectively. The cells were blocked and incubated with a primary monoclonal antibody against SREBP-1 (BioVision Inc., 1:100 dilution). After incubation with fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific Inc.), cells were counterstained with DAPI (Invitrogen, Thermo Fisher Scientific Inc). After washing with PBS, cells were examined using an FV10i confocal laser microscope (Olympus) or the ImageXpress® Micro XLS Widefield High-Content Analysis System (Molecular Devices). Data analysis was performed using the MetaXpress software package (Molecular Devices).
4
Quantitative High-Content Imaging Cytotoxicity
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Cytotoxicity and mitochondrial integrity were quantitatively assessed by high-content imaging after conclusion of Ca2+ flux measurements at 90 min and prior to TempO-seq lysate preparation at 24 hrs as described previously (Grimm et al., 2016 (link)). Briefly, cell culture medium including (90 min) or excluding (24 hrs) Ca2+ dye was replaced with 25 μl of staining solution (2 μg/ml Hoechst 33342 and 0.2 μM MitoTracker Orange in iCell cardiomyocyte maintenance medium). Following 15 min incubation at 37°C and 5% CO2 the staining solution was discarded and replaced with an equal volume of fresh maintenance medium. Images were then acquired using an ImageXpress Micro Confocal cellular imaging system (Molecular Devices), using DAPI and Cy3 filters for Hoechst 33342 and MitoTracker Orange, respectively. Images were processed using the instrument-specific MetaXpress software package (Molecular Devices). Quantification of imaging-based parameters for concentration-response assessment was achieved using the multi-wavelength cell scoring application module.
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