To detect the proliferation of cultured HUVECs after stimulation, EdU (1 μM) was added to the culture medium 4 h before the end point of 24 h stimulation. EdU staining was then performed according to the manufacturer’s instructions of EdU Apollo488 In Vitro Kit (Ribobio) and analyzed by High-Content Imaging System (ImageXpress Micro, Molecular Device, USA).
Imagexpress micro
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro is a high-performance, automated, widefield microscopy system designed for high-content imaging and analysis. It captures high-quality images across multiple samples and channels, enabling quantitative data collection for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Metaxpress software, by Molecular Devices (11 mentions)
Hoechst 33342, by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Merck Group (8 mentions)
Metaxpress, by Molecular Devices (7 mentions)
Alexa fluor 488 anti rabbit antibody, by Thermo Fisher Scientific (4 mentions)
Microtest 96 well plate, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Vybrant dyecycle green, by Thermo Fisher Scientific (3 mentions)
Acumen ex3, by SPT Labtech (3 mentions)
Metaxpress analysis software, by Molecular Devices (3 mentions)
Hoechst 33342, by Molecular Devices (2 mentions)
Tcs sp2 aobs, by Leica (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Matrigel, by Corning (2 mentions)
Carboxy h2dcfda am, by Thermo Fisher Scientific (2 mentions)
Anti phospho myosin light chain 2 ser19, by Cell Signaling Technology (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Hcs nuclearmask blue stain, by Thermo Fisher Scientific (2 mentions)
140 protocols using imagexpress micro
1
Quantifying HUVEC Proliferation via EdU
2
High-Throughput RNA Interference Screening
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Double-stranded RNAs (250 ng/well) were pre-spotted in 384-well plates (Ambion AM8500). DL1 cells stably maintaining “pMtnA eGFP MALAT1” were grown for one passage in the absence of hygromycin B. A total of 15,000 cells were then seeded in each well of the 384-well plates in 10 µL of serum-free Schneider's Drosophila media. After 1 h, complete media (with serum) was added (20 µL/well). Cells were grown for 3 d and then treated with 10 µL of media containing CuSO4 (500 µM final concentration, Fisher BioReagents BP346-500) for 6 h. Cells were fixed (5% formaldehyde, Fisher BioReagents BP531-500) and stained with Hoechst 33342 (Sigma B2261) to visualize nuclei. Four images per well (eGFP and Hoechst 33342 staining) were captured at 20× magnification using an automated microscope (ImageXpress Micro, Molecular Devices) and analyzed using MetaXpress software. “Mean stain integrated intensity” of eGFP and “total cell number” were calculated for each image, and the median and interquartile ranges (IQR) were used to calculate a z-score for each plate: (Mean stain integrated intensity-median)/(IQR*74). Wells with robust Z-scores ≥1.3 or ≤ −1.3 were considered hits and Gene Ontology (GO) analysis was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID), v6.8 (https://david.ncifcrf.gov/home.jsp ) with standard parameters.
3
Visualizing Mycobacterial Infection in BMDMs
4
Immunofluorescence Microscopy Protocol
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Cells were fixed in 4% formaldehyde in PBS for 30 minutes at RT, washed with PBS followed by 15 minutes of 0.2% Triton X-100 permeabilization on ice. 3% BSA in 1xPBS was used for blocking 30 minutes at RT. Primary antibodies were added overnight at 4°C (Hoechst and fluorescent secondary antibodies were added post washing for 1 to 2 hrs at RT). Images were taken either with a 20X air objective, an automated inverted epifluorescence microscope (ImageXpressMicro, Molecular Devices), with a 40X or 100X oil objective on a confocal microscope (Leica TCS SP2 AOBS or 3i imaging system). See section on Fixed/live cell imaging for more detailed description. See Supplementary Table 5 for antibody information.
5
Automated High-Content Screening of DNA Damage Response
6
Quantifying Neurite Outgrowth in Neurons
7
High-Throughput Cell Cycle Analysis
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Example 11
HeLa cells were plated in 384-well plates (1500 cells/well) and treated with 10 μM drugs for 20 hours. Cells were fixed and stained with 5 μM Vybrant DyeCycle Green (Invitrogen) for 1 hour at room temperature and plates were scanned with an Acumen eX3 (TTP Labtech) fluorescence cytometer using its 488 nm laser and a cell cycle histogram profile was generated for each well. For the G2/M secondary screen, 20 hours post drug addition cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS and stained with Alexa-488-phospho-histone-H3 (Ser10, Cell Signaling) and 1 μg/ml Hoechst 33342 for 1 hour. Plates were imaged with an ImageXpress Micro (Molecular Devices) high-content fluorescence microscope. Data analysis was performed using the CDD (Collaborative Drug Discovery) software and outputs were exported to Excel. The quality of the screen was assessed by calculating the Z′ factor (Z′ factor=1-3×(σp+σn)/(|μp−μn|)), which takes into account the dynamic range of the assay and variance of the data. (Zhang J. H., et al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999, 4(2): 67-73.) The screen performed with an average plate Z′ factor of 0.51±0.09, within the optimal performance range of 0.5-1.47 (Zhang, op. cit.)
8
Quantifying HeLa Cell Detachment
9
Evaluating Crry/p65 Functional Decrease
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To evaluate the functional decrease in Crry/p65 in RCB1994 cells, we measured the deposition area of C3c on these cells induced by normal mouse serum (MS). RCB1994 cells were seeded at 5.0 × 104 cells·well−1 and incubated with RPMI medium (control) or calf thymus histones (200 μg·mL−1) for 30 min at 37 °C. After incubation, the cells were washed twice with PBS, then incubated with 5% normal MS or heat‐inactivated MS for 1 h at 37 °C. Then, the cells were fixed with 4% paraformaldehyde phosphate buffer solution for 10 min at room temperature. The cells were washed twice with PBS, and then, the cells were incubated in the dark with FITC‐conjugated rabbit polyclonal IgG anti‐C3c containing Hoechst 33342 solution for 30 min at room temperature. Heat‐inactivated MS was prepared by incubation for 30 min at 56 °C. To measure the C3c‐positive area per cell, we counted cells and measured the positive area in five randomly selected fields, with images being captured at 200× magnification under a BZ‐X700 Fluorescence Microscope (Keyence). Measurements of the C3c‐positive area in images were performed using ImageXpress MICRO (Molecular Devices, San Jose, CA, USA).
10
High-Content Screening of Schistosomula Phenotypes
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The larval High-Content Screening (HCS) was run as described previously [31 (link)]. Briefly cultured schistosomula were cultured for 72 hours in absence or presence of compound. After a gentle re-suspension using a Biomek FxP to generated an even distribution of parasites across the well, images were collected at 10x for phenotype analysis and at 4x over a time-series for motility analysis using an Image Xpress micro (Molecular devices, USA). Image analysis was then conducted using Pipeline Pilot 9.0 (Biovia, USA). Phenotype analysis was conducted using a previously validated two class Laplacian-modified Bayesian categorization model built using standard schistosomacides and motility analysis by individual parasite area displacement over the time series [31 (link)]. To generate larval IC50s the percent inhibition at specific compound concentrations were calculated by comparison to the mean DMSO scores (32 wells) on each plate. These were then imported into Prism 6 (Graphpad) to generate IC50 values.
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