MDA-MB231 and ELK3KD-231 cells were seeded into 96-well plates (2×104 cells per well) and co-cultured for 4 hours with NK92MI cells labeled with CellTrace far-red reagent (Invitrogen). Then, 5 µM CellEvent caspase-3/7 green detection reagent (Invitrogen) was added to the culture medium. After 2 hours, fluorescence images of caspase activity were obtained using the ImageXpress Imaging System (Molecular Devices, San Jose, California, USA).
Imagexpress imaging system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress imaging system is a high-content screening platform designed for automated cellular imaging and analysis. It captures and analyzes images of cells or other biological samples in multiwell plates. The system provides quantitative data on various cellular parameters, such as morphology, protein expression, and cell behavior.
Automatically generated - may contain errors
Lab products found in correlation
Acetic acid, by Thermo Fisher Scientific (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Kmno4, by Merck Group (2 mentions)
Ag325, by Merck Group (2 mentions)
Celltrace far red reagent, by Thermo Fisher Scientific (1 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vehicle dimethylsulfoxide, by Merck Group (1 mentions)
5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions)
96 well imaging plate, by BD (1 mentions)
Methanol, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
6 protocols using imagexpress imaging system
1
NK92MI-Mediated Apoptosis Assay
2
Fluoro-Jade C Staining of Neurodegeneration
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Neurodegeneration was assessed using FJC (AG325, MilliporeSigma; Burlington, MA, USA) staining. Brain slices were thawed on ice, then incubated in 0.06% (w/v) KMnO4 (Sigma) in distilled H2O (dH2O) for 10 min, rinsed 3x in dH2O, and incubated in 0.00015%, w/v FJC and 0.5 μg/mL DAPI (Invitrogen; Carlsbad, CA, USA) in 0.1% acetic acid (Acros Organics; Geel, Belgium) in dH20 for 10 min in the dark. Slides were then dipped in xylene (X5SK-4, Assay grade; Thermo Fisher Scientific) for 1 min, mounted in Permount (Thermo Fisher Scientific), coverslipped, and imaged at 20X magnification on a high content ImageXpress imaging system (Molecular Devices; Sunnyvale, CA, USA). Neurodegeneration of the cortex and hippocampus was evaluated by counting neurons positively labeled with FJC (per mm2) at 7 days post-exposure, as described previously (Zolkowska et al., 2012 (link)).
3
Fluoro-Jade C Staining of Neurodegeneration
4
Immunofluorescent Staining of Neuronal Markers
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Staining was performed at room temperature. A blocking buffer was first introduced for 1 h followed by incubation with primary antibody overnight in 4 °C. Neurons were stained using mouse anti-MAP-2 antibody, clone HM-2 (dilution 1:1000; Sigma-Aldrich, catalog number M4403, species mouse). Primary antibody was visualized with Alexa Fluor 488 or 546-conjugated goat anti-mouse secondary antibody (dilution 1:250, Invitrogen, Burlington, Canada). Cell nuclei were stained with Hoechst S769121 (nuclear yellow). Cells were stored in 4 °C in the dark before imaging.
Images were taken using the automated ImageXpress® imaging system (Molecular Devices, Sunnyvale, CA) through a 10× objective microscope lens, displaying 4 or 9 sites per well. Images were analyzed with the software MetaXpress® (Molecular Devices, Sunnyvale, CA) using the algorithm “multiwavelength cell scoring”50 (link). Cells were defined according to fluorescence intensity and size at different wavelengths. Data from all sites per well were averaged to one data point.
Images were taken using the automated ImageXpress® imaging system (Molecular Devices, Sunnyvale, CA) through a 10× objective microscope lens, displaying 4 or 9 sites per well. Images were analyzed with the software MetaXpress® (Molecular Devices, Sunnyvale, CA) using the algorithm “multiwavelength cell scoring”50 (link). Cells were defined according to fluorescence intensity and size at different wavelengths. Data from all sites per well were averaged to one data point.
5
Vitamin D Modulates Myoblast Proliferation
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For EdU incorporation, mitotic G2 and cell viability analysis, myoblasts were seeded in 96-well glass bottom microplates (BD Falcon) at a density 1.5 × 103 cells/well. After 48 h, media was replaced containing 0.01, 1, 100 nM 1α,25(OH)2D3 (vitamin D; Sigma-Aldrich) or vehicle (dimethyl sulfoxide) (Sigma-Aldrich) in either low- or high-serum media and stimulated for 24 and 48 h. Following stimulation myoblasts were incubated with 10 μM EdU 60 min prior to fixation in 4% paraformaldehyde (PFA) in PBS for 15 min at RT. Fixed cells were washed with PBS and permeabilised with 0.3% Triton X-100 in PBS (PBST) and then incubated 30 min at room temperature with 2 μM Alexa 647-conjugated Azide, 10 mM ascorbic acid, 2.5 mM CuSo4 in PBS. DAPI staining was performed by a 1-h incubation at 4°C with 10 μg/mL RNAse, followed by 10 min of 1 μg/mL DAPI in PBS at room temperature. All plates were imaged on an ImageXpress imaging system (Molecular Devices) at 10× magnification, and segmentation of nuclei and measurement of fluorescence intensities were performed in CellProfiler (29 (link)).
6
Quantifying Cell Cycle Dynamics with EdU Labeling
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To label the cells in S phase, cells in passage 4 or 5 were plated at a density of 3,000 cells per well in a 96-well imaging plate (BD Falcon, Franklin Lakes, NJ) and cultured for one day prior to pulse with 1 μM 5-ethynyl-2′-deoxyuridine (EdU; Molecular Probes, Eugene, OR) for 1 h. The cells were fixed using 3.7% formaldehyde (Sigma Aldrich, St Louis, MO) for 5 min and permeabilised in −20 °C methanol (Sigma Aldrich) for 2 min followed by incubation with DAPI. For the click-chemistry reaction, cells were incubated in a solution containing 100 mM Tris (pH 6.8), 1 mM CuS04, 100 mM ascorbic acid and azid fluorophore (#A10277, Invitrogen, Carlsbad, CA) for 1 h at room temperature. Images were acquired on an ImageXpress imaging system (Molecular Devices, Sunnyvale, CA) using a Nikon Plan Fluor ELWD 20 × 0.45 NA objective, resulting in 640 × 479 pixel 16-bit images. Image analysis was performed using CellProfiler36 (link). Background was subtracted using the CellProfiler inbuilt illumination function with 120-pixel block size. Nuclei were segmented based on DAPI intensity using a manually-set threshold and the integrated intensity of DAPI and EdU fluorescence was measured.
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