For EdU staining, cells were pulsed for 30 min with 10 μM EdU from the Click-iT Edu Alexa Fluor 488 imaging kit (ThermoFisher). Cells were then fixed by 4% PFA/PBS for 15 min, and permeabilized with 0.25% Triton/PBS for 15 min. The Click-It reaction was then performed according to manufacturer’s instructions. Immunostaining was carried out as described. Images were acquired on a fully automated Molecular Devices ImageXpress Micro microscope and intensity analysis was performed using MetaXpress quantification software.
Imagexpress micro microscope
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro is a high-content imaging system designed for automated, quantitative analysis of cellular and subcellular features. It provides high-resolution, high-speed image acquisition and analysis capabilities for a wide range of applications in life science research.
Automatically generated - may contain errors
Lab products found in correlation
Wellmate, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (2 mentions)
Eclipse ti2 microscope, by Nikon (2 mentions)
F1141, by Merck Group (2 mentions)
P96 1.5h n, by Cellvis (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Cyquant direct cell proliferation assay, by Thermo Fisher Scientific (1 mentions)
Synergy neo, by Agilent Technologies (1 mentions)
Phospho kap1, by Fortis Life Sciences (1 mentions)
Phospho p53, by Cell Signaling Technology (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Mts assay, by Promega (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Brdu antibody, by Cell Signaling Technology (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Cls3814, by Corning (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
21 protocols using imagexpress micro microscope
1
EdU-based Cell Proliferation Assay
2
High-Content Analysis of MSC-ALL Co-Cultures
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Co-cultures of MSC and ALL cells were stained with CyQUANT® Direct Cell Proliferation Assay (Life Technologies). The mixture of component A (diluted to 1:1200 in AIM-V medium) and component B (diluted to 1:80 in AIM-V medium) was added to the cells for 60 minutes at 37°C and 5% CO2 prior imaging. Images were taken on the ImageXpress Micro microscope (Molecular Devices) with a CoolSNap HQ camera (Photometrics) and a 0.3 NA 10x Plan Fluor objective (Nikon). Image acquisition was set to cover more than 50% of each well in a total of 9 images (Fig. 2B ).
For analysis, images were first segmented using the CellProfiler software [19 (link)] and distinction of viable ALL cells from dead ALL cells as well as MSC was implemented in a machine learning tool for high-content analysis (Advanced Cell Classifier, ACC) [20 (link)]. An ‘a trous’ wavelet transform-based spot detection algorithm [18 ] was built into the CellProfiler framework. For each detected cell, over 50 intensity and texture-based features were extracted. The list of extracted features, CellProfiler pipelines, and the parallelization code are accessible atwww.highcontentanalysis.org . For correct assignment of ALL cells, the open source Advanced Cell Classifier program [20 (link)] was used with the random forest classification method for all experiments [40 ].
For analysis, images were first segmented using the CellProfiler software [19 (link)] and distinction of viable ALL cells from dead ALL cells as well as MSC was implemented in a machine learning tool for high-content analysis (Advanced Cell Classifier, ACC) [20 (link)]. An ‘a trous’ wavelet transform-based spot detection algorithm [18 ] was built into the CellProfiler framework. For each detected cell, over 50 intensity and texture-based features were extracted. The list of extracted features, CellProfiler pipelines, and the parallelization code are accessible at
3
Identifying Selective Inhibitors of p97 in the UPS
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A dual-reporter stable HeLa cell line was generated to monitor UPS function in mammalian cells31 (link). A ubiquitin fusion degradation (UFD) reporter UbG76V-GFP and an oxygen-dependent degradation domain of HIF1α fused to luciferase (ODD-Luc). We could identify molecules that selectively block p97 activity but no other general components of the UPS by seeking compounds that stabilize UbG76V-GFP but not ODD-Luc. Cells were seeded on 384-well plates (3000 cells/well) and grown for 18 h. Cells were treated with modified DMEM (without phenol red, folic acid, riboflavin, and vitamin B12) containing MG132 (4 μM) for 1 h and washed twice with pre-warmed PBS. Modified DMEM containing FBS (2.5%), CHX (50 μM), and DMSO or a test compound (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μM) was added to each well. Plates were imaged on the ImageXpress Micro microscope (Molecular Devices) after 160 min. Luciferase activity were measured by adding D-Luciferin (10 μl of 2.5 mg/mL in PBS) into each well containing 38 μl of medium and incubated at room temperature for 10 min. Use Synergy Neo (BioTek) to read Luciferase signal.
4
Quantitative Analysis of DNA Damage Response
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Fibroblasts were transfected with 200 nM of ASOs as described above. Forty-eight hours after transfection, cells were irradiated with 1.5 Gy using a caesium-137 source, and then incubated for 60 min at 37 °C. Cells were washed in PBS, fixed in 4% (w/v) paraformaldehyde and permeabilized with 0.1% (w/v) Triton X-100 in PBS at room temperature. Cells were then incubated overnight in PBS with 3% BSA and antibodies to phospho-P53 (Cell Signaling Tech) and phospho-KAP1 (Bethyl Lab) and were visualized with immunoglobulin G Alexa Fluor conjugates (Life Technologies). DNA was counterstained with Hoechst 33342. Images were collected with the ImageXpress Micro microscope (Molecular Devices) and processed with MetaXpress (Molecular Devices). The abundance of targets expressed in nuclei was quantified.
5
Measuring Protease-Mediated Protein Degradation
6
siRNA Screen for IAV Host Factors
7
Chemotaxis Assays for MDA-MB-231 Cell Migration
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Chemotaxis assays were performed to assess MDA-MB-231 cell migration upon αvβ3 integrin blocking by DisBa-01. For transwell assays, a 24-well insert, ThinCert™ translucent PET membrane, 8.0 μm pore (Greiner Bio-one®, Frickenhausen, Germany) were used. MDA-MB-231 cells (1 × 10 5) in medium without serum were treated with DisBa-01 for 30 min at room temperature and inserted into the upper part of the Boyden chamber. The lower chamber contained medium plus 10% SFB. The system was incubated for 16 h (MDA-MB-231 cells) or 24 h (HUVEC) at 37 °C in normoxic and hypoxic conditions. Filters were fixed with 3.7% paraformaldehyde and the remaining cells on the upper surface were removed using a cotton swab. The nuclei of migrating cells were stained with 0.7 ng/µL DAPI solution (Thermo Fisher Scientific, Waltham, MA, USA, Catalog Number: 62248). Membranes were assembled on a microscope slide for automated cell counting in an ImageXpress Micro microscope (Molecular Devices, San Jose, CA, USA) under 10× magnification with the Meta-X-press software, and quantified using the Multi Wavelength Cell Scoring.
8
Measuring Cell Proliferation via Multiple Assays
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Cell proliferation was measured using three different methods including MTS assay (Promega, Madison, WI, USA), BrdU ELISA assay (Cell Signaling, Danvers, MA, USA) and nuclei counting. Primary NP cells were seeded into laminin coated 96-well plates at a density of 4 × 103 cells/well (1.25 × 104 cells/cm2). Cells were treated with indicated compound or DMSO control for 72 hours before the assays. For MTS assay 20 μl reagent was added to each well and incubated for 3 hours at 37 °C before the absorbance at 490 nm (A490) was measured. For BrdU assay the cells were chased with 10 μM BrdU in the last 4 hours and then fixed, stained with BrdU antibody and then HRP-conjugated secondary antibody, and developed with TMB substrate to measure A450. For nuclei counting the fixed cells were stained with Hoechst 33342 dye (Life Technologies, Grand Island, NY, USA). The plate was scanned with an ImageXpress Micro microscope (Molecular Devices, Sunnyvale, CA, USA) using a 4× objective to take an image covering the whole well for each well. Cell numbers were counted using the ImageJ software with an automatic nuclei counter plug-in.
9
Comprehensive Cell Proliferation and Survival Assays
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For the cell proliferation assay, ReNCell VM cells were seeded into laminin coated 96-well plates at a density of 4 × 103 cells/well (1.25 × 104 cells/cm2). Cells were treated with indicated compound or DMSO control for 48 hours before they are fixed and stained with Hoechst 33342 dye (Life Technologies, Grand Island, NY, USA). The plate was scanned with an ImageXpress Micro microscope (Molecular Devices, Sunnyvale, CA, USA) using a 4× objective to take an image covering the whole well for each well. Cell numbers were counted using the ImageJ software with an automatic nuclei counter plug-in. Cell proliferation was also measured by BrdU assay. Cells were grown in laminin coated 96-well plates, and treated with indicated compound for 48 hours. After that cells were pulsed with 10 μM BrdU for 2 hours, then stained with BrdU antibody (1:1000, Cell Signaling, Danvers, MA, USA). For the survival assay, cells were differentiated in the presence of indicated compounds. Then the cells were fixed, permeabilized and assayed with the Roche in situ Cell Death detection kit following the manufacturer’s instruction. Cells were also blocked and stained with additional markers as indicated.
10
Mammosphere Formation and Propagation
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Cells were plated as single cells at a density of 5 × 102 viable cells/well in ultralow attachment six-well plates (Corning, CLS3814). Cells were grown in a serum-free DMEM or phenol-red free DMEM, supplemented with B27 (Invitrogen, 17504-044), 20 ng/mL EGF (Sigma, E9644) and 20 ng/mL bFGF (R&D systems, 233-FB-025). Mammospheres were grown for 10–14 days, and phase contrast images were obtained using the ImageXpress Micro microscope (Molecular Devices). For the second-generation experiment, first-generation mammospheres were collected from multiple wells and spun at 500 × g per 5 min. The pellet was resuspended in 50 μl of Trypsin, and the sample was passed 25 times through a sterile needle to get single-cell suspension. The same density of cells as in first-generation culture was seeded, and cells were allowed to grow for 14 days.
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