Adipogenic staining was performed as described previously (Eom et al., 2018 (link)). In brief cells were plated in a 96-well plate in 200 μl ASC media (DMEM/F12 media (Life Technologies) supplemented with 10% FBS, 1% GlutaMax (Life Technologies, Auckland) and 1% penicillin/streptomycin (Life Technologies) and cultured at 37°C, 5% CO2. On day four, 100 μl of media was replaced with adipogenic differentiation media (ASC media with 1 µM dexamethasome, 10 µM insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 200 µM indomethacin (all from Sigma Aldrich, Auckland) and standard ASC media was added to control wells. Half media changes were performed every 3 days until day 14. Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes®) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes®). Fluorescent images were taken using the ImageXpress Micro XLS high content screening system (Molecular Devices™). Nine images were taken per well at 10 × magnification and quantitative data was generated using the MetaXpress v 5.3.0.1 (Molecular Devices™) software.
Imagexpress micro xls high content screening system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro XLS is a high-content screening system that captures and analyzes images of cells or other samples. It is designed to provide high-resolution imaging and data analysis capabilities for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc pi apoptosis detection kit, by BD (1 mentions)
2 protocols using imagexpress micro xls high content screening system
1
Adipogenic Differentiation Quantification
2
Erianin-Induced Apoptosis in HeLa Cells
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For apoptotic cells analysis, HeLa cells were treated with various concentrations of erianin (3.9, 7.9, 15.7, 31.4) µM and 0.05 µM PTX for 12, 24, 36 and 48 h. The cells were then collected and washed with ice-cold PBS. Next, early apoptosis was measured using an Annexin V-Fluorescein Isothiocyanate (FITC)/PI apoptosis detection kit (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA). The cells were suspended with 100 µl of binding buffer and stained with 5 µl annexin V-FITC and 5 µl PI for 20 min at room temperature in the dark and then 400 µl binding buffer was added. The proportion of apoptotic cells were determined by flow cytometry and observed using the ImageXpress® Micro XLS High Content Screening system (Molecular Devices, LLC, Sunnyvale, CA, USA), with Hoechest 33342 staining.
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