DRG cultures were prepared as described previously [9 (link)–11 (link)]. 8-10 week old C57BL/6J mouse DRGs were dissociated and cultured for 24 hours in 96-well PDL plates (BD BioCoat, Corning 354461) coated with laminin (Sigma L2020, 10 μg/ml in PBS, one hour at room temperature) or CSPG (Millipore, CC117). CSPG (3.3 ng/well in 50 μl PBS) was immobilized by drying overnight in a tissue culture hood and then rinsed with 50 μl of water to remove excess salt. DRG neurons were plated at a density of 2,500 neurons/cm2 (833 neurons/well). Small molecules were added to the culture medium at the time of cell plating. After 24 hours of axon outgrowth, cultures were fixed and immunostained for βIII-tubulin (Sigma-Aldrich, T8660, 1:800) with an Alexa Fluor 488 secondary antibody (Invitrogen, A-11029, 1:500). Images were acquired using an ImageXpressmicro High Content Imaging System (Molecular Devices) with a 10X objective (four sites per well). Axon outgrowth was quantified using MetaXpress (Molecular Devices). The total axon length per neuron (axon length) is reported. n = 12-96 wells per condition.
Imagexpress micro high content imaging system
Manufactured by Molecular Devices
The ImageXpress Micro High-Content Imaging System is a fully automated, high-throughput microscope designed for live-cell and fixed-sample screening applications. It features advanced optics, a high-resolution camera, and a user-friendly software interface to capture and analyze images of cells and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Metaxpress software, by Molecular Devices (2 mentions)
Metaxpress high content image acquisition and analysis, by Molecular Devices (2 mentions)
Cc117, by Merck Group (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Dnase, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Acuityxpress software package, by Molecular Devices (1 mentions)
96 well woundmaker, by Sartorius (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
10 protocols using imagexpress micro high content imaging system
1
DRG Neuron Axon Outgrowth Assay
2
High-Content Imaging of Cellular Responses
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Four sites (each imaged at two excitation wavelengths of 405 and 561 nm) per well were acquired sequentially at 10 × magnification on an ImageXpress Micro High-Content Imaging System from Molecular Devices. Images were analyzed using MetaXpress software (Molecular Devices). For treated cells, total cell count and total m-Cherry intensity above background were determined using the “TransFluoHT” module and average m-Cherry signal per cells of four sites were calculated for each well. For transfected cells, the percentage of cells with positive nuclear-localizing mCherry signal above background was determined using the “Translocation-Enhanced” module. The average fold-change percentage positive mCherry cells for each sample well was normalized with reference to mean of siNT (n = 8) in the same plate. Raw data were processed and analyzed using the ScreenSifter22 software, and the average ± SD for screen replicates were determined. The threshold for positive hits (> 2) was derived using the derivative method available in the ScreenSifter software. Similarly, the average cell count per sample well was normalized to the mean cell count in siNT wells in the same plate, with the threshold arbitrarily set to 80%.
3
High-Content Imaging of Stem Cell Clusters
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Automated image acquisition and analyses were conducted as described previously using the ImageXpressmicro high content imaging system (Molecular Devices, Sunnyvale, CA) [33 (link)]. The system is equipped with an incubator to maintain a constant temperature, carbon dioxide concentration, and humidity during analyses. On the day of analysis at the end of a designated culture period, culture plates were transferred to our institutional imaging facility and placed in the incubator of the image capture apparatus. For each well of the plates, 30 pictures were acquired; the incubator was set to 37 °C and maintained at 5% CO2 during the procedure. Thereafter, MetaXpress software (Molecular Devices) was used to determine the number and surface area of clusters. Guided by GFP signals, clusters were defined as objects with the minimum width of 30 µm and the maximum width of 180 µm, which correspond to the aggregates of at least six cells [33 (link)]. Cluster surface area was defined as the total area of all pixels inside projected perimeter of cluster that have fluorescence intensity above the set threshold value (local background 150 gray levels). Cluster numbers represent those of SSCs that survived while cluster area reflects the activity of SSCs to produce committed progenitors and the ability of progenitors to proliferate during the exposure to compounds [33 (link)].
4
High Content Microscopy Protocol
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High content microscopy was performed as previously described (Hari & Acosta, 2017 ). Where included, cells were treated with 50 mM BrdU (Sigma, 858811) for 16 hr prior to fixation. Briefly, cells were fixed with 10% formalin for 10 min, permeabilized with 0.2% Triton/PBS for 10 min and then blocked with blocking solution (1% BSA/0.2% fish gelatin in PBS) for 1 hr. Primary antibody diluted in blocking solution was then added, and the cells were incubated for 1 hr at RT. Anti‐BrdU solution was supplemented with 0.5 U/μl DNAse (Sigma D4527) and 1 mM MgCl2. Incubation with fluorescent secondary antibodies for 1 hr and 1 μg/ml DAPI for 30 min followed. The cells were visualized using confocal microscopy. 96‐well plates were scanned using an ImageXpress Micro High Content Imaging System (Molecular Devices), acquiring multiple images. Automated analysis of these images was performed using the software MetaXpress 5.1.0.46 (Molecular Devices).
5
High-Content Screening of Protein Localization
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High Content screening was performed using the ImageXpress micro high content imaging system (Molecular Devices, Downingtown, PA) driven by MetaXpress software. Six images per well were acquired in each of three channels (DAPI, FITC, TRITC) at 10X magnification in an unbiased fashion. Images were analyzed using the MetaXpress ‘Multiwavelength Scoring’ (for mCherry-NLS signals) and ‘Transfluor’ modules (for DLK-GFP signals). Data were exported to a spreadsheet using the AcuityXpress software package (Molecular Devices).
6
In Vitro Wound Healing Assay
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The wound-healing assay was performed using the protocol suggested by the manufacturer (Sartorius). Briefly, A549 NucLight Red cells were seeded in 96-well plates (Greiner, Cat# 655087) at 40000 cells/well in RPMI 1640 media supplemented with 10% FBS and 1% antibiotics. After 24 h, a wound scratch was created in the confluent cell monolayer using a 96-well woundmaker (Sartorius), followed by washing three times with PBS. Then, the medium was replaced with RPMI 1640 medium without FBS. TGFβ (10 ng/ml), GA (5 μM), or GNA (5 μM) was added at the indicated concentrations. After 24 h of treatment, the plates were imaged using the ImageXpress Micro High-Content Imaging System (Molecular Device). The wound-healing activity was quantified by measuring the width (W) of the wound using ImageJ and expressed as (W0h—W24h)/W0h × 100.
7
Genome-wide siRNA Screening in PTEN-Isogenic Cells
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We carried out the primary siRNA screen in PTEN+ and PTEN- MCF-10A cell lines. Cell lines were screened using the whole-genome siGENOME siRNA library from Dharmacon (now Thermo-Fisher). The entire library, comprising 21,121 siRNA SMARTpools (pools containing 4 different siRNAs targeting each gene) was arrayed (one SMARTpool per well) in 68 black-walled 384-well plates (Greiner, #781091). Three types of control were included on all screening plates: non-silencing negative controls (48 wells containing Qiagen AllStars Negative Control siRNA; SI03650318), essential gene-positive controls (8 wells containing a siRNA pool targeting PLK1), and PTEN-SSL gene-positive controls (8 wells containing a siRNA pool targeting CDC25A). The screen was done by reverse transfecting 250 cells with 25 nM siRNA and 0.25 μl HiPerFect transfection reagent (Qiagen) in each well and then culturing the cells for 5 days. After 5 days, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton-x, and stained with Hoechst 33,342 (Molecular Probes). Stained nuclei were imaged and automatically counted using an ImageXpress Micro high-content imaging system (Molecular Devices) to generate raw cell counts after siRNA perturbation.
8
High-Content Quantification of Neurite Outgrowth
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For neurite and axon outgrowth, images from 48- to 60-h DRG or cortical neuron cultures were acquired using the ImageXpress Micro High-Content Imaging System (Molecular devices). These images were analyzed for neurite outgrowth using WIS-Neuromath (Rishal et al., 2013 (link)). Neurites were visualized using NF immunofluorescence. Neurite lengths were measured for at least 75 neurons per treatment group per culture preparation.
9
Measuring Intracellular Potassium in MAYV-Infected BMDMs
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Intracellular concentration of potassium was determined by fluorescence emission of asante potassium green-2 (APG-2, TEFLabs). BMDMs (2×104) were seeded in black, clear-bottom 96-well plates, and treated with PAM(3)CSK(4) for 4 hours, then infected with MAYV (MOI of 5). After 2 hours of infection, BMDMs were incubated with 5 μM APG-2 in RPMI without FBS and phenol red for 30 min. The cells were washed with PBS, and RPMI without phenol red was replaced. Nine images per well were recorded at 40× magnification with the ImageXpress Micro High-Content Imaging System and processed with MetaXpress High-Content Image Acquisition and Analysis (Molecular Devices).
10
Intracellular K+ Measurement in B. Abortus-Infected BMDMs
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To increased extracellular [K+] assay, BMDMs were seeded at a density of 5 × 105 cells/well in 24-well. We incubated BMDMs in a medium containing 80 mM KCl 1 h before infection. Then, BMDMs were infected with B. abortus at an MOI of 100 in the same medium for 17 h and IL-1β was measured in the supernatant. Intracellular concentration of K+ was determined by fluorescence emission of Asante Potassium Green-2 (APG-2, TEFLabs, Austin, EUA). Briefly, BMDMs (2 × 104) were seeded in black, clear-bottom 96-well plates, infected with B. abortus at an MOI of 100. After 6 h of infection, cells were incubated with 5 μM APG-2 in RPMI without FBS and phenol red for 30 min. BMDMs were washed with PBS, and the media was replaced with RPMI without phenol red. Four images per well were recorded at 40× magnification with the ImageXpress Micro High-Content Imaging System and processed with MetaXpress High-Content Image Acquisition and Analysis (Molecular Devices). The images were analyzed using ImageJ, and the concentration of intracellular K+ in each cell was calculated as a percentage: MFI540nm (inquired cell)/ Σ MFI540nm (control cells) ×100.
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