To confirm the presence of PPARγ protein in situ across treatments, cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 as described above for either 2, 4, 6, 8, or 24 h. At exposure termination, cells were fixed with 4% formaldehyde at room temperature for 10 min. Cells were then rinsed three times with 1X phosphate-buffered saline (PBS) and incubated in blocking buffer [1X PBS + 0.1% Tween-20 (PBST), 2 mg/mL bovine serum albumin, and 2% sheep serum] at room temperature for 1 h by shaking gently. Blocking buffer was then replaced with a 1:100 dilution of a human PPARγ-specific antibody (E-8, sc-7273; Santa Cruz Biotechnology, Dallas, TX USA) diluted in blocking buffer and allowed to incubate overnight at 4 °C. Cells were then incubated with a 1:500 dilution of Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (ThermoFisher Scientific, Waltman, MA USA) overnight at 4 °C. Cells were then counterstained with a 1:3 solution of DAPI Fluoromount-G (Southern Biotechnology, GA) for 5 min, rinsed with 1X PBS three times, and then imaged (at 10X magnification) and analyzed using our ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA USA).
Imagexpress micro xls widefield high content screening system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro XLS Widefield High-Content Screening System is a high-performance imaging system designed for cell-based assays and high-content screening applications. The system utilizes widefield optics to capture images of cells or samples across a large field of view, enabling efficient and comprehensive data acquisition. The system is engineered to provide reliable and reproducible results, supporting a range of cell types and experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Dapi fluoromount g, by Southern Biotech (4 mentions)
Metaxpress 6, by Molecular Devices (3 mentions)
Alexa fluor 488 conjugated goat anti mouse igg1 antibody, by Thermo Fisher Scientific (2 mentions)
Corona green am, by Thermo Fisher Scientific (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Metaxpress high content image acquisition and analysis software, by Molecular Devices (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
13 protocols using imagexpress micro xls widefield high content screening system
1
Quantifying PPARγ Protein Expression in Treated Cells
2
High-Content Screening of EGFP-Infected Cells
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ImageXpress® Micro XLS Widefield High-Content Screening System (Molecular Devices, San Jose, CA, USA) was used to visualize and quantify the number of EGFP-infected cells. Per each well, one image, covering 5.32% of the well surface, was captured using two fluorescent channels, i.e., DAPI and GFP. Images were then analyzed with MetaXpress software. To detect and analyze EGFP-infected cells, two settings were fixed: the typical cell area and the GFP expression. Cell area was first determined by the presence of nuclei based on DAPI stained images, and with several manual delimitations of cell edges based on bright field images. Upon first identification, the number of cells expressing EGFP was determined.
3
Quantifying FASN Protein Levels In Situ
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To quantify FASN protein levels in situ, cells were exposed to either vehicle (0.1% DMSO), TBBPA, or TCBPA as described above for 24 h. At exposure termination, cells were fixed with 4% formaldehyde at room temperature for 10 min. Cells were then rinsed three times with 1× phosphate-buffered saline (PBS) and incubated in blocking buffer [1× PBS + 0.1% Tween-20 (PBST), 2 mg/mL bovine serum albumin, and 2% sheep serum] at room temperature for 1 h by shaking gently. Blocking buffer was then replaced with a 1:500 dilution of a human FASN-specific antibody (G-11, sc-48357; Santa Cruz Biotechnology, Dallas, TX, USA) in blocking buffer and allowed to incubate overnight at 4 °C. Cells were then incubated with a 1:500 dilution of Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Thermo Fisher Scientific, Waltman, MA, USA) overnight at 4 °C. Cells were then counterstained with a 1:4 solution of DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) for 5 min, rinsed with 1× PBS three times, and then imaged (at 10× magnification) and analyzed using our ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA, USA).
4
Quantifying Neutral Lipid Abundance in HepG2 Cells
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To determine whether exposure to TBBPA or TCBPA affected neutral lipid abundance, HepG2 cells were exposed to vehicle (0.1% DMSO), TBBPA, or TCBPA as described above and then stained for neutral lipids using Oil Red O (ORO) (MilliporeSigma, St. Louis, MO, USA) as previously described (Cheng et al., 2021 (link)). Briefly, cells were fixed using 4% paraformaldehyde for 20 min at room temperature. Cells were then rinsed with 60% isopropanol and stained with ORO working solution (1.8 mg ORO per 1 mL 60% isopropanol) for 10 min at room temperature. Cells were then rinsed four times with molecular biology-grade water for 5 min at room temperature. After the final wash, cells were counterstained with a 1:4 solution of DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) for 5 min at room temperature. Cells were washed three more times with molecular biology-grade water and then imaged (at 10× magnification) and analyzed using our ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA, USA).
5
Automated High-Content Neurite Outgrowth Analysis
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Neurite outgrowth was evaluated using the ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices, LLC., San Jose CA, USA) with a 10× objective. This system automatically focuses on and scans the fields of individual wells. Fluorescence images were obtained using a high-resolution camera and matched excitation filters for channels 1 (blue; Hoechst 33342) and 2 (green; βIII-tubulin). The measured object was counted as a neuron cell if it had valid measurements, including size, shape, and intensity. The resulting fluorescence images were analyzed to obtain various parameters, such as total neurite outgrowth, total nuclei, total processes, and total branches, using the MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices, LLC.).
6
Quantifying Embryonic Ionocytes in Zebrafish
7
Quantifying Neutral Lipid Accumulation in HepG2 Cells
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To determine whether exposure to ciglitazone or GW 9662 affected neutral lipid abundance, HepG2 cells were stained for neutral lipids using Oil Red O (ORO) (Sigma-Aldrich, St. Louis, MO, USA) following exposure to vehicle (0.1% DMSO), ciglitazone, or GW 9662 as described above. Briefly, cells were fixed using 4% paraformaldehyde for 20 min at room temperature. Cells were then rinsed with 60% isopropanol and stained with ORO working solution (1.8 mg ORO per 1 mL 60% isopropanol) for 10 min at room temperature. Cells were then rinsed four times with molecular biology-grade water for 5 min at room temperature. After the final wash, cells were counterstained with a 1:3 solution of DAPI Fluoromount-G (Southern Biotech) for 5 min at room temperature. Cells were washed three more times with molecular biology-grade water and imaged (at 10X magnification) and analyzed using our ImageXpress Micro XLS Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA USA).
8
Titration of Recombinant Virus by EGFP Imaging
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The titration of VSV*ΔG-Luc(VSV-G) was performed by two-fold serial dilution on monolayers of Vero E6 cells. After 1 h of infection, fresh medium was added, and cells were incubated overnight at 37 °C. Infection was quantified by measuring EGFP-positive infected cells per well site using ImageXpress® Micro XLS Widefield High-Content Screening System (Molecular Devices). Data were analyzed with MetaXpress and GraphPad Prism 7 softwares.
9
Influenza Infection Screening in A549 Cells
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A549 cells were seeded at a density of 8,000 cells per well in a 96-well assay plate and transduced with RNAi lentiviruses; shRNA-positive cells were selected using puromycin (3 μg/ml). At 5 days posttransduction, the cells were infected with strain A/WSN/33 (WSN) for 6 h and harvested for immunofluorescence staining using anti-NP antibody. The images were captured using the ImageXpress Micro XLS wide-field high-content screening system (Molecular Devices).
10
Quantifying Embryonic Sodium Levels
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