Cells were fixed in 4% polyformaldehyde for 10 min at room temperature, treated with 1% Triton X-100 in PBS for 10 min, blocked with 5% bovine serum albumin (BSA) for 1 hr, and hybridized to a mouse monoclonal antibody against the P16 protein (Ventana Roche Diagnostics, E6H4, Switzerland) overnight at 4°C. Samples were incubated with FITC-labeled secondary antibody (KPL, 172–1806, USA) for 1 hr at room temperature, followed by nuclear staining with DAPI. Fluorescence images were acquired and analyzed with an ImageXpress Micro High Content Screening System (Molecular Devices, USA).
Imagexpress micro high content screening system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro High Content Screening System is a fully automated, high-throughput imaging platform designed for a wide range of cellular assays. It features high-resolution optics, multiple fluorescence channels, and advanced image analysis capabilities to enable quantitative measurements of cellular morphology, protein expression, and other cellular parameters.
Automatically generated - may contain errors
Lab products found in correlation
Metaxpress image analysis software, by Molecular Devices (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
96 well microtiter plate, by Greiner (3 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Cell scoring metaxpress software, by Molecular Devices (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Multi wavelength cell scoring application module, by Molecular Devices (2 mentions)
Fluoview fv10i, by Olympus (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Arvo mx, by PerkinElmer (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Necrostatin 1, by Abcam (1 mentions)
Z vad fmk, by Abcam (1 mentions)
Ab2386, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Pdgf bb, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Viewplate, by PerkinElmer (1 mentions)
Cellrox detection reagent, by Thermo Fisher Scientific (1 mentions)
25 protocols using imagexpress micro high content screening system
1
Immunofluorescence Analysis of P16 Protein
2
Quantitative Immunocytochemistry of RNA-Binding Proteins
3
Fungal Interaction with Host Cells
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HC cells were seeded onto a 35-mm glass-based dish (2 × 105 cells) and incubated at 37 °C with 5% CO2 overnight. The next day, cells were co-incubated with different strains of fungi (5 × 106 cells) for 8 hours. ROS production was analyzed based on the incorporation of CM-H2DCFDA (final concentration, 5 µM) (Life Technologies, Eugene, OR, USA) for 15 minutes at 37 °C, and cells were immediately monitored for their fluorescein isothiocyanate (FITC) fluorescence signals using confocal microscopy or an ImageXpressMICRO High Content Screening System (Molecular Devices). The morphological analysis was performed using MetaXpress Image Analysis software (Molecular Devices).
4
Intracellular ROS Quantification
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ROS production was analyzed based on the incorporation of the chloromethyl derivative of 2′,7′‐dichlorodihydrofluorescein diacetate (CMH2DCFDA; Life Technologies) (2.5 μm ) for 30 min at 37 °C. After chemical treatment for the indicated time, the cells were monitored for their FITC fluorescence signals using a plate reader (ARVO MX; Perkin Elmer Inc.) or an ImageXpressMICRO High‐Content Screening System (Molecular Devices).
5
Cell Proliferation Assay with 5-FU
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Cells at 5000 per well were plated in 96-well plates for 24 h, and then treated with 10− 5 M 5-FU for 48 h. After incubation with 10 μM EdU for a further 2 h, the cells were fixed in 4% paraformaldehyde (PFA) and stained with Click-iT reaction solution at 100 μL per well. Hoechst 33342 was used to stain the nuclei. Images were obtained using an ImageXpress Micro High Content Screening System (Molecular Devices, USA) at 200× magnification, and the ratio of EdU/Hoechst 33342-double-positive cells was quantified.
6
Measuring Intracellular ROS Levels
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The cells were fluorescently stained for intracellular ROS status by incubating them in 5 µM CellROX Deep Red detection reagent (Life Technologies) for 30 min, followed by three washes in PBS. After nuclear staining with 40 µg Hoechst 33342 dye (Dojin, Kumamoto, Japan)/ml for 5 min, images were captured to detect the intracellular ROS signal using the ImageXpress MICRO high content screening system (Molecular Devices, Sunnyvale, CA, USA). Cell fluorescence was quantified using the accompanying software.
7
Measuring Spindle Orientation in U2OS Cells
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U2OS cells were seeded at 3,000 cells per well in 96‐well plates with L‐shaped micropatterns (CYTOO) at a density of 15,000 cells/ml. Prior to seeding, plates were coated with 20 μg/ml fibronectin (Sigma) for 2 h at RT. Following seeding, cells were imaged every 5 or 10 min for up to 24 h at 37°C in a 5% CO2 environmental chamber using an ImageXpress Micro High Content Screening System (Molecular Devices Inc.). To measure spindle orientation in subconfluent cultures, cells were seeded at 50% confluency, grown overnight and imaged as previously described 60 . Spindle angles were measured using a vector drawn through the division axis at anaphase bisecting a vector drawn through the cell's long axis determined prior to prophase.
8
Viability Assay for Lipid-Treated Mesenchymal Stem Cells
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LMSCs were plated at a density of 1.5 × 103 cells/cm2 in 96-well Black Flat Bottom Polystyrene Microplates. After 24 h, the cells were pre-treated for 3 h with 20 µM Z-VAD-FMK (BioVision, Milpitas, CA, USA), 10 mM 3-methyladenine (BioVision), or 100 µM necrostatin-1 (ABCAM, Cambridge, UK), or for 4 h with 4 mM N-acetyl-l-cysteine (NAC; Eurofarma, SP, Brazil). The media was then changed and 7-KC added at different concentrations for 24 h. LMSCs without pre-treatment were used as controls. Cells were then incubated with 0.1 µg/mL Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and 0.5 μL propidium iodide (PI) (Molecular Probes) for 15 min. The ImageXpress Micro High Content Screening System (Molecular Devices) was used to determine the number of live and dead cells. Nine sites per well and three wells per treatment were acquired. Cell Scoring MetaXpress software (version 5, Molecular Devices) was used to analyze the number of cells and the viability as previously described [36 (link)].
9
High-Content Screening of Cellular Markers
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Cells were seeded in Greiner 96‐well microtiter plates (Greiner Bio‐One), fixed in 4% paraformaldehyde for 10 min, and then incubated in 0.5% Triton X‐100 in PBS for 10 min at room temperature as previously reported 7 . After the cells were blocked in 5% fetal bovine serum/TPBS for 1 h at room temperature, they were incubated overnight at 4 °C with primary antibodies, including mouse anti‐TG2 (1 : 200, ab2386; Abcam, Cambridge, MA, USA), rabbit anti‐nuclear factor erythroid‐2‐related factor 2 (Nrf2) (1 : 400, ab62352; Abcam), mouse anti‐Ki67 (1 : 200, 350502; BioLegend, San Diego, CA, USA), or control rabbit/mouse IgG. The cells were then washed and stained with fluorochrome (FITC/TRITC)‐conjugated secondary antibodies (1 : 500; Invitrogen, Eugene, OR, USA) for 20 min at room temperature. Cell nuclei were visualized using DAPI (1 : 2000; Wako Industries). Cellular fluorescence signals were detected using an ImageXpressMICRO High‐Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using the ‘Multi‐Wavelength Cell Scoring Application Module’ in MetaXpress Image Analysis software (Molecular Devices).
10
Immunofluorescence Quantification of Orm1 in Primary Mouse HPCs
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Primary mouse HPCs were isolated as described above and seeded in Greiner 96-well microtiter plates (Greiner Bio-One, Monroe, NC, USA) at a concentration of 1.5 × 105 cells/mL. The cells were treated with PDGF-BB (PeproTech, Rocky Hill, NJ, USA, #100-14B, 20 ng/mL) in DMEM containing 2% FBS for 48 h. Then, the cells were fixed in 4% paraformaldehyde (PFA) for 10 min and incubated with 0.1% Triton X-100 in PBS for 5 min at room temperature. After blocking with 5% FBS-PBS for 1 h at room temperature, cells were incubated with rabbit anti-mouse Orm1 antibody (1 μg/mL; PAA816Mu01; Cloud-Clone Corp) or control rabbit IgG overnight at 4 °C. The cells were then washed and stained with donkey anti-rabbit Cy5-conjugated secondary antibody (1:500, Invitrogen), while nuclei was visualized by Hoechst staining (Wako Industries). Immunofluorescence staining signals were detected with a Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss Inc.) or an ImageXpressMICRO High Content Screening System (Molecular Devices, Sunnyvale, CA, USA) and morphological analysis was performed using MetaXpress Image Analysis software (Molecular Devices).
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