The 384-well screening plates were imaged in an automated fashion using the Molecular Devices ImageXpress microscope. Robotic plate handling was used to load and unload the plates. The objective used for acquisition was a 10X S Fluor with 0.45NA. 9 sites per well were imaged in a 3x3 grid without spacing or overlap of the images with three channels for monitoring the cell's nuclei (DAPI stain), the cell's actin cytoskeleton (DY-547-phalloidin) and the GFP-expressing bacteria (pM975).
Imagexpress microscope
Manufactured by Molecular Devices
The ImageXpress microscope is a high-performance imaging system designed for a variety of cell-based assays. It captures high-quality images and data efficiently, enabling researchers to gain deeper insights into biological processes.
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Lab products found in correlation
Celltracker dye, by Thermo Fisher Scientific (1 mentions)
Axiovert 100 microscope, by Zeiss (1 mentions)
Magnafire ccd camera, by Optronics (1 mentions)
Prism 8, by GraphPad (1 mentions)
Dexamethasone (dex), by Selleck Chemicals (1 mentions)
Abt 263, by Selleck Chemicals (1 mentions)
24 well plate, by Corning (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Fluoromyelin, by Thermo Fisher Scientific (1 mentions)
10 protocols using imagexpress microscope
1
High-Content Imaging of Bacterial Infection
2
Quantifying Oligodendrocyte Morphology
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WT and TMEM10 Oli-sneu cells were cultured under proliferation conditions for 48 hrs. Cells were then fixed with 4% PFA and stained with Cell Tracker dye (ThermoFisher). Images were taken with an ImageXpress microscope (Molecular Devices) and analyzed using MetaXpress software. Measurements taken included process outgrowth; number of branches per cell; number of processes per cell; and percentage of cells exhibiting process outgrowth. For morphological analysis of primary OPCs, cell cultures were fixed in 4% PFA and immunolabeled for MBP. Images were taken using an Axiovert 100 microscope (Carl Zeiss) with a MagnaFire CCD camera (Optronics). 50–100 cells were randomly selected and individually outlined using the selection tool in ImageJ41 (link). Mean fluorescence intensity and cell area were measured and multiple shape descriptors calculated using ImageJ. Circularity is defined as the degree to which a particle is similar to a circle, taking into consideration the smoothness of the perimeter and solidity as an inverse measure of the overall concavity of a particle42 .
3
Drug Sensitivity and Synergy Profiling in ALL
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Drug sensitivity and drug synergy profiles were evaluated using ALL cells in co-culture with MSCs as described above. A Tecan D300 robot was used to dispense drugs at the indicated dilutions. ABT-263 (navitoclax) and dexamethasone (Selleckchem) concentrations for synergy were based on the half maximal inhibitory concentration (IC50) of the different samples screened. Cell viability was evaluated after 72 h of drug treatments using CyQuant staining and imaging-based cell viability analysis on an ImageXpress microscope (Molecular Devices; ref. 15 (link)). All the dose response curves and the IC50 were represented and calculated using GraphPad Prism 8. The synergy was calculated using SynergyFinder tool21 (link),22 (link). The resulting ZIP score indicates synergism (ZIP score ≥ 1), additivity (ZIP score 0–1) or antagonism (ZIP score ≤ 0), and is here represented by a 3D graph21 (link),22 (link).
4
Microglia Phagocytosis of Glioma Cells
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Fluorescent beads phagocytosis assay was employed to analyze live microglia phagocytosis in co‐cultures with glioma cells. OAβ42 in simple F12 medium was thawed on ice and polymerized in a 24‐well plate (Corning) at 37 °C. GL261 was labeled with GFP. BV2 was unstained and replicated by staining with CellTracke Red CMTPX (Yeasen). The plates were imaged for 10 h at the basetime of addition with lower concentration (1 μM), higher concentration (5 μM) OAβ42, or vehicle in a motorized ImageXpress microscope (Molecular Devices). Image sequences were combined with the ImageXpress, and quantification was analyzed with ImageJ. Only when the GFP‐labeled cell interacts with the unstained cell a phagocytosis issue can be counted. And the phagocytosis index was identified as the reduced percentage of these GFP‐labeled cells from the base‐time to the ending time.
5
Flow Cytometric Sorting and Fitness Assay
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Cells were analyzed at a medium flow rate in a FACSaria flow cytometer. The signal coming from cell aggregates was eliminated on the basis of the forward scatter area and height. Populations were separated on the basis of fluorescence intensity. We collected ∼106 events for each sample. Immediately after sorting, cells were imaged under fluorescence light or incubated at 30°C for fitness assays, either in an Infinite M200 microplate reader (Tecan) or in an Image Xpress Microscope (Molecular Devices).
6
Cell Density and Proliferation Assay
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Wild type (WT) and TMEM10 Oli-neu cells were plated at low cell density (1300 cells/cm2) and cultured under normal conditions for 2, 24, 48 and 72 hrs. At each time point, cells were fixed with 4% paraformaldehyde (PFA) and stained with the nuclear marker DAPI (4’,6-diamidino-2-phenylindole). Images were taken with an ImageXpress microscope (Molecular Devices) and the number of DAPI-positive nuclei counted using MetaXpress software.
7
Immunocytochemistry of Human Stem Cells
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Adult human SCs and fibroblasts were grown to 80% confluence, washed with sterile PBS and then fixed in 4% paraformaldehyde (PFA) for 5 min. Cells were then permeabilized using 0.5% Triton X-100 and 5% BSA for 1 h at room temperature. Primary antibodies (Table 1 ) were incubated at 4°C overnight. Anti-Nestin (NES) (for SC cultures) or anti-FN1 (for fibroblast cultures) antibodies were used in all cases, as a costain to validate within each culture that the appropriate cell type of interest was indeed present. The next day, unbound primary antibodies were washed then incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 1 h or/and Fluoromyelin (1:200, Life Technologies), washed, and nuclei were counterstained with Hoechst (1:2000, Sigma) and mounted with Permafluor mounting media (for confocal imaging) or left in PBS (for ImageExpress imaging). For quantification, cells were imaged and quantified using an ImageXpress Microscope (Molecular Devices). Each condition consisted of cells from three to four donors, with three to four technical replicates (consisting of 12 images/replicate), per cell type per antibody.
8
High-Throughput Contractility Screening of HLF
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For all screening runs, HLF cryopreserved at passage 3 were thawed into a T75 flask 7 days before the experiment. Screening runs comprised of 2 days and were executed with an integrated automation platform comprising of a Multidrop for plate-wise reagent and cell addition, a Biomek FXP for pinning drug solution, a Cytomat automated incubator and robotic arm for transporting plates between incubation and imaging stations, and a Molecular Devices ImageXpress microscope for highthroughput automated imaging.
On day 1, FLECSplates were filled with media, equilibrated to RT, given drug from the library plates (250nL of 1 mM drug solution yielding a 3.5 µM final concentration), then shaken on a plate shaker for 15 min. Next, HLF were dissociated and seeded into drug-bearing FLECSplates as previously described with TGF-β1 in plate columns 2-23 and without TGF-β1 in columns 1 and 24. On day 2, following 24 h of incubation, cells were given Hoechst 33,342 live nuclear stain as previously described and the plates were imaged with a 4x objective at one position per well capturing both the micropatterns (TRITC channel) and stained nuclei (DAPI channel). Images were subsequently downloaded in .TIFF format and analyzed using Forcyte's proprietary computer vision to derive quantitative contractility data.
On day 1, FLECSplates were filled with media, equilibrated to RT, given drug from the library plates (250nL of 1 mM drug solution yielding a 3.5 µM final concentration), then shaken on a plate shaker for 15 min. Next, HLF were dissociated and seeded into drug-bearing FLECSplates as previously described with TGF-β1 in plate columns 2-23 and without TGF-β1 in columns 1 and 24. On day 2, following 24 h of incubation, cells were given Hoechst 33,342 live nuclear stain as previously described and the plates were imaged with a 4x objective at one position per well capturing both the micropatterns (TRITC channel) and stained nuclei (DAPI channel). Images were subsequently downloaded in .TIFF format and analyzed using Forcyte's proprietary computer vision to derive quantitative contractility data.
9
Automated Microscopy for Cellular Imaging
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Microscopy was performed with Molecular Devices ImageXpress microscopes. A MetaXpress plate acquisition wizard with no gain, 12-bit dynamic range, and 9 sites per well in a 3 × 3 grid were used with no spacing and no overlap and laser-based focusing. A 4′,6′-diamidino-2-phenylindole (DAPI) channel was used for imaging nuclei, a green fluorescent protein (GFP) channel for bacteria, and a red fluorescent protein (RFP) channel for F-actin or dsRed of bacteria in the entry assay. Robotic plate handling was used to load and unload plates (Thermo Scientific). The objective was a 10× S Fluor with 0.45 numerical aperture (NA). The Site Autofocus was set to “All Sites,” and the initial well for finding the sample was set to “First well acquired.” Z-Offset for Focus was selected manually, and manual correction of the exposure time was applied to ensure a wide dynamic range with low overexposure. Images from the different siRNA screens are available upon request.
10
High-Content Microscopy Imaging Protocol
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Microscopy was performed with Molecular Devices ImageXpress microscopes. We used the MetaXpress plate acquisition wizard with no gain, 12 bit dynamic range, 9 sites per well in a 3×3 grid with no spacing and no overlap and laser-based focusing. Channels were assay specific (see Additional file
1 : Table S2). Robotic plate handling was used to load and unload plates (Thermo Scientific). The objective was a 10X S Fluor with 0.45NA. The Site Autofocus was set to “All Sites” and the initial well for finding the sample was set to “First well acquired”. Z-Offset for Focus was selected manually and “AutoExpose” was used to get a good exposure time. Manual correction of the exposure time was applied to ensure a wide dynamic range with low overexposure, when necessary.
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