Cells were plated in 96 well clear-bottom plates. Experiments with siRNA were performed 72 hours post-transfection while experiments with overexpression plasmids were performed 48 hours post-transfection. Cells were incubated with media containing 10uM EdU for 30 minutes, pre-extracted for 5 minutes on ice in 20mM HEPES, pH 7.0, 50mM NaCl, 3mM MgCl2, 300mM sucrose, and 0.5% Triton X-100 followed by fixation in 3% paraformaldehyde. Cells were blocked for 1 hour in PBS containing 1mg/mL BSA, 5% goat serum, 0.1% Triton X-100, and 1mM EDTA. EdU was labeled by addition of 2mg/mL sodium ascorbate, 2mM copper sulfate, and 5uM Alexa Fluor conjugated azide in PBS for 30 minutes. Primary antibody incubation was performed for 1 hour in 1% BSA in PBS followed by a 45-minute incubation in secondary antibody. Nuclei were stained with a 5-minute incubation with DAPI in PBS. Plates were imaged on a Molecular Devices ImageXpress system. The integrated nuclear intensity of PCNA of EdU positive cells was analyzed. In overexpression experiments, cells were co-transfected with 0.1ug of a vector expressing GFP-H2B to visualize transfected cells and the integrated nuclear intensity of PCNA in EdU and GFP positive cells was analyzed.
Imagexpress system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress system is a high-content screening platform that enables automated image acquisition and analysis. It provides automated fluorescence microscopy capabilities for a wide range of applications, including cell-based assays, phenotypic screening, and high-throughput imaging.
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Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Incucyte flr imaging system, by Sartorius (1 mentions)
Spark 20m, by Tecan (1 mentions)
Anti neurofilament 200, by Merck Group (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
96 well imaging plate, by BD (1 mentions)
Dmi6000 imaging system, by Leica camera (1 mentions)
Xf base medium minimal dmem, by Agilent Technologies (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Facs aria 3 system, by BD (1 mentions)
12 protocols using imagexpress system
1
EdU Incorporation Assay for Proliferation
2
Culturing Primary Neurons for Neurite Outgrowth Analysis
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Primary neurons were cultured from postnatal day 1 rat pups following procedures previously described [72 (link)]. Briefly, the pup brain was removed after decapitation, was minced by scissors, and trypsinized by 0.25% trypsin (Thermo 25200056) incubation for 30 min at 37 °C. Next, the lysates were centrifuged and filtered through a 40 μm nylon mesh (Falcon 352340, Corning, NY, USA). The isolated cells were seeded onto poly-D-lysine (Sigma P6407, Fremont, CA, USA)-coated dishes and cultured in a basal neural medium (Thermo 21103-049) containing 2% B27 neural supplements (Thermo 17504044). The cultured neurons performed neuritis outgrowth along the developmental days and were subjected to oxygen and glucose deprivation (OGD) at day 9 while they displayed matured pattern of neuritis complexity. We screened MAP2 and NeuN double-positive cells by the ImageXpress System (Molecular Devices, Sunnyvale, CA, USA) to quantify the neurite outgrowth. The Neurite Outgrowth Module of the MetaXpress software (Molecular Devices) was used to analyze the number of neurons, length of total outgrowth per cell, and the number of processes and branches per cell.
3
DNA Damage Response Imaging Assay
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Cells were plated in 96-well clear-bottom plates, incubated with media containing 10 μM EdU for 20 min, pre-extracted for 5 min on ice in 20 mM HEPES, pH 7.0, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100 followed by fixation in 3% paraformaldehyde. The cells were blocked for 1 h in PBS containing 5% BSA. A click chemistry reaction was completed by the addition of 2 mg/ml sodium ascorbate, 2 mM copper sulfate, and 5 μM Alexa Fluor 647 conjugated azide in PBS for 30 min. Anti-γH2AX antibody incubation was performed for 1 h in 1% BSA in PBS followed by a 45-min incubation in secondary antibody. Nuclei were stained with a 5-min incubation with DAPI in PBS. Plates were imaged on a Molecular Devices ImageXpress system, and integrated nuclear intensity of γH2AX in EdU-positive cells was quantitated using the Molecular Devices software.
4
Multiparametric Cell Viability Assay
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Unless stated otherwise, cells were seeded at 1,000 cells/well into flat-bottom 96-well plates (Greiner 655180). Edge wells were excluded and plates were left for 30 min at room temperature before transferring to an incubator. Plates for real-time analysis were transferred to the IncuCyte FLR imaging system (Essen BioScience) as previously described (10 (link)). Unless stated otherwise, compounds were added (5 μL/well) at two days post seeding, for five days. Compounds were reconstituted in dimethyl sulfoxide (DMSO). Plates were assayed for cell viability using the resazurin reduction assay and/or cell counts as described previously (10 (link)). The Spark 20M (TECAN) was used to quantify resazurin fluorescence following best practice procedures described previously (11 (link)). The ImageXpress system (Molecular Devices) was used to image Hoechst 33342 stained plates. CellProfiler (www.cellprofiler.org ) was used to enumerate cell counts as previously described (12 (link)). Normalized cell viability was calculated as follows: sample/DMSO × 100. Relative half-inhibitory concentrations (EC50) were calculated in Prism from the nonlinear regression algorithm of the normalized cell viability plotted against an 8-point compound dilution.
5
Immunofluorescence Analysis of Umbelliprenin Effects
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Cells were cultured in plates and treated with Umbelliprenin (0, 6, 12, 24 μM for AGS cells and 0, 12.5, 25, 50 μM for BGC-823 cells) for 24 h. The treated cells were fixed with 4% paraformaldehyde for 10 min and washed twice with PBS. These cells were blocked with 5% bovine serum albumin in PBS for 60 min, and then incubated with the primary antibody (diluted 1:200 in PBS containing 3% BSA) overnight at 4°C. After washing twice with PBS, the cells were co-incubated with the IgG PE-conjugated secondary antibody (diluted 1:400 in PBS containing 3% BSA) and DAPI for 1 h at room temperature. The cells were examined with the Image Xpress system (Molecular Devices, USA).
6
Quantifying DNA Damage in Proliferating Cells
7
EdU Incorporation Assay for Proliferation
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Cells were plated in 96 well clear-bottom plates. Experiments with siRNA were performed 72 hours post-transfection while experiments with overexpression plasmids were performed 48 hours post-transfection. Cells were incubated with media containing 10uM EdU for 30 minutes, pre-extracted for 5 minutes on ice in 20mM HEPES, pH 7.0, 50mM NaCl, 3mM MgCl2, 300mM sucrose, and 0.5% Triton X-100 followed by fixation in 3% paraformaldehyde. Cells were blocked for 1 hour in PBS containing 1mg/mL BSA, 5% goat serum, 0.1% Triton X-100, and 1mM EDTA. EdU was labeled by addition of 2mg/mL sodium ascorbate, 2mM copper sulfate, and 5uM Alexa Fluor conjugated azide in PBS for 30 minutes. Primary antibody incubation was performed for 1 hour in 1% BSA in PBS followed by a 45-minute incubation in secondary antibody. Nuclei were stained with a 5-minute incubation with DAPI in PBS. Plates were imaged on a Molecular Devices ImageXpress system. The integrated nuclear intensity of PCNA of EdU positive cells was analyzed. In overexpression experiments, cells were co-transfected with 0.1ug of a vector expressing GFP-H2B to visualize transfected cells and the integrated nuclear intensity of PCNA in EdU and GFP positive cells was analyzed.
8
Automated Neurite Outgrowth Analysis
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Fixed DRG neurons were incubated in blocking solution (5% donkey serum, 0.3% Triton X-100/1× PBS) for 30 min, primary antibodies in diluent (0.1% Triton X-100, 0.1% BSA, 0.04% EDTA/1× PBS) for 1 h, washed twice in PBS, secondary antibodies in diluent for 1 h, Hoechst-33258 (14530, Sigma, 1:500 in PBS) for 10 min, and finally washed twice in PBS. All steps were performed at RT. Primary antibodies used were a cocktail of anti-Neurofilament200 (mouse, N5389, Sigma-Aldrich, 1:800) and anti-Tubulin β3 (mouse, 801213, Bio Legend, 1:1,000) to facilitate automated neurite analysis. The secondary antibody used was Alexa Fluor 488 donkey anti-mouse (A21202, Invitrogen, 1:200). Each well was imaged at 10× using the ImageXpress system (Molecular Devices) and a composite was stitched using Microsoft Image Composite Editor (Microsoft). The composite images were each divided into nine images of equal size and all analyzed using the MetaXpress (Molecular Devices) automated neurite outgrowth function.
9
Immunofluorescence Imaging of Claudin-2 in CHO-K1 Cells
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Cells were seeded at a density of 1x105 cells/ml in 96 well plates (3904, Costar) and grown at 37°C, 5% CO2 until around 70% confluence. CHO-K1 cells were transiently transfected with GFP-claudin-2 as described earlier. Cells were fixed for 20 minutes at room temperature in 10% neutral buffered formalin solution before being thoroughly washed in PBS. Then cells were stored at 4°C until required for staining.
For immunofluorescence, cells were permeabilised with 0.1% Triton/PBS for 10 minutes. Primary antibodies were applied at a dilution 1:500 for 1 hour at room temperature, before washing in PBS/0.1% Triton-X100. Appropriate secondary antibodies were applied at a dilution of 1:500 for 1 hour at room temperature (donkey anti-rabbit pAb IgG DyLight 594nm (ab96921, Abcam) or goat anti mouse IgG AlexaFluor 546 (A11030, Life Technologies)). Cells were washed twice in PBST, and once in PBS before counterstaining with Hoechst 33342 (H3570, Life Technologies) at a dilution of 1:5000 in PBS for 10 minutes. After 2 further PBS washes, images were read on an ImageXpress system (Molecular Devices, US).
For immunofluorescence, cells were permeabilised with 0.1% Triton/PBS for 10 minutes. Primary antibodies were applied at a dilution 1:500 for 1 hour at room temperature, before washing in PBS/0.1% Triton-X100. Appropriate secondary antibodies were applied at a dilution of 1:500 for 1 hour at room temperature (donkey anti-rabbit pAb IgG DyLight 594nm (ab96921, Abcam) or goat anti mouse IgG AlexaFluor 546 (A11030, Life Technologies)). Cells were washed twice in PBST, and once in PBS before counterstaining with Hoechst 33342 (H3570, Life Technologies) at a dilution of 1:5000 in PBS for 10 minutes. After 2 further PBS washes, images were read on an ImageXpress system (Molecular Devices, US).
10
Live-Cell Imaging and FRET Microscopy
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For live-cell imaging, cells were seeded in 96-well imaging plates (BD Falcon) using Leiboiwitz-15 medium (Invitrogen) and followed on an ImageXpress system (Molecular Devices) using a 20× NA 0.45 objective or on a Leica DMI6000 Imaging System using a 20× NA 0.4 objective. Images were processed and analysed using ImageJ. Nuclei and cytoplasm were selected by manual drawing. FRET microscopy was performed as in Hukasova et al (2012) (link) and simultaneous monitoring of FRET and a PCNA chromobody was performed as in Akopyan et al (2014) (link).
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