Uptake studies were conducted with HIFU-treated DiO’-labeled liposomes (NUSL, USL, DiO’ at 0.5% mol of total lipid) that had been loaded with AF647-labeled ML1. Liposomes were diluted 1:10 in OptiMEM (final concentrations 2 g/ml AF647-ML1 and 7 mM total lipid) and treated with HIFU as described above with the following acoustic settings: 5 MPa for 1 min exposure and 24 MPa for 1 min exposure. Samples were transferred to the ice bath and used for uptake studies without further processing. Before adding the samples, nuclei of CT26 cells were pre-stained with Hoechst 33342. After replacement of the culture medium with the HIFU-treated samples, 96-well µClear® black plates (Greiner) were transferred into a Yokogawa Cell Voyager CV7000s microscope (Tokyo, Japan). Live-cell confocal microscopy images were taken for 4 h at 37°C and analyzed for uptake of liposomes (red), uptake of ML1 (green), and nuclei (blue). Uptake was semi-quantified with Columbus® image analysis software (PerkinElmer) using automated protocols for nuclei and cytoplasm detection and build-in functionalities for fluorescence intensity determination.
Columbus image analysis software
Manufactured by PerkinElmer
Sourced in United States
Columbus image analysis software is a comprehensive platform for high-content imaging and analysis. It provides tools for image acquisition, management, and quantitative analysis of cellular and subcellular features. The software is designed to handle a wide range of imaging data and deliver robust and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Operetta, by PerkinElmer (2 mentions)
Operetta high content imaging system, by PerkinElmer (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Clear black plates, by Greiner (1 mentions)
Cell voyager cv7000s microscope, by Yokogawa (1 mentions)
Mitotracker green, by PerkinElmer (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Operetta instrument, by PerkinElmer (1 mentions)
96 well microplate, by PerkinElmer (1 mentions)
Glomax discover instrument, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Concanavalina 488, by Thermo Fisher Scientific (1 mentions)
Phalloidin 547, by Thermo Fisher Scientific (1 mentions)
Image express microscope, by Molecular Devices (1 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Hcs cell mask deep red, by Thermo Fisher Scientific (1 mentions)
Anti p21 antibody, by Abcam (1 mentions)
18 protocols using columbus image analysis software
1
Liposome Uptake Kinetics by HIFU
2
Mitochondrial Membrane Potential Assay
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Measurement of mitochondrial membrane potential was determined using the potential-sensitive dye Tetramethylrhodamine ethyl ester perchlorate (TMRE; Molecular Probes). Briefly, 2000-3000 cells were cultured in a 384-well microplate for 48 hours then incubated for 30 minutes in DMEM containing 20 nM MitoTracker Green FM (Molecular Probes). The cells were pre-incubated with 20 μM Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; to eliminate mitochondrial membrane potential) or solvent control at 37 °C for 1 hour prior to adding TMRE. After washing with media, 25 nM TMRE in media containing 0.5 μg/mL Hoechst 33342 (Molecular Probes) was added to the cells and incubated for 1 hour at 37 °C. TMRE and MitoTracker Green fluorescence intensities of the mitochondria in live cells were imaged using the Opera automated confocal microscope and analyzed with the Columbus Image Analysis Software (Perkin Elmer).
3
Automated Cell Viability Assay
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Cells were seeded in 96-well microplates (PerkinElmer); the seeding cell confluency was specifically optimized for each cancer cell line to have cells in a growth phase at the end of the assay. After overnight incubation at 37 °C, cells were treated with DMSO (Merck) for the negative control and with five concentrations of selected drugs in triplicate. Cells were then incubated at 37 °C for 72 h. Cell viability was assessed by measuring either luminescence with GloMax® Discover instrument from Promega or by nuclei count using the Operetta instrument from PerkinElmer. Luminescence measurements were normalized using background wells as manufacturer protocol. For luminescence measurement, cells were treated with Promega CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer protocol. For nuclei count, cells were washed with PBS 1×, fixed with paraformaldehyde (PFA) 4% for 10 min at room temperature, washed with PBS 1×, incubated at room temperature in the dark with HOECHST 33342 (Thermo Fisher Scientific) diluted 1:1000 in PBS 1× for 10 min and finally washed with PBS 1×. Nuclei count was performed using Columbus image analysis software (PerkinElmer). All drugs used in this study were purchased from Selleckchem.
4
Multiparametric Imaging of Organelle Morphology
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The hTERT RPE-1 cells and NGLY1−/−, PMM2F119L/−, and DPAGT1+/− mutant lines were plated in 384 well plates at a density of 3000 cells per well. The next day compounds were added to the cells at a final concentration of 10 µM and incubated for 24 h. Cells were stained with MitoTracker Red (Molecular Probes, cat. M7512) mitochondrial stain for 30 min according to the manufacturer’s protocol, then media was removed, cells were fixed in 4% PFA/PBS, permeabilized with 0.1% NP-40, and blocked with 3% BSA/ PBS overnight. For staining, ConcanavalinA-488 (Molecular Probes, cat. C11252), phalloidin-547 (Molecular Probes, cat. A22283), and DAPI were added to the wells, then washed before imaging. Images were acquired on a Molecular Devices Image Express microscope at 4 fields per well. Feature extraction from images was done with Perkin-Elmer Columbus Image Analysis software, and feature analysis and hit determination was performed using TIBCO Spotfire analysis package.
5
Ectodermal Lineage Differentiation of Pluripotent Stem Cells
6
Quantifying Viral Envelope Protein Expression
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Cells were then processed for immunofluorescent detection of E1 envelope glycoprotein with Cy3-labeled antibody as previously described (Rouille et al., 2006 (link)). Nuclei were stained with 1 μg/mL DAPI. Confocal images were recorded on an automated confocal microscope IN Cell Analyzer 6000 (GE Healthcare Life Sciences) using a 20X objective with exposure parameters 405/450 nm and 561/610 nm. Six fields per well were recorded. Each image was then processed using the Columbus image analysis software (Perkin Elmer). Nuclei were first segmented and cytoplasm region extrapolated based on the DAPI staining. Then a cell was considered “infected” for a high ratio of the intensity of the Cy3 staining in the cytoplasm region (relative to that of the nucleus) and Cy3 intensity above a fixed threshold. Quantity of cells per well and MOI were calculated to obtain no more than 40% of infected cells 30 h post infection, allowing the automated quantification. Data were then normalized to DMSO control set-up at 100% of infection.
7
Immunofluorescent Staining of p21 Protein
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Cells were permeabilised by the addition of 100 µl of 0.1% (v/v) Triton X-100 in PBS per well for 20 min. Thereafter, the cells were blocked by incubation for 1 h with 50 µl blocking buffer (5% (w/v) BSA in PBS) followed by incubation overnight at 4 °C with 50 µl of anti-p21 antibody (Abcam, cat. no. Ab109520) diluted 1:1000 in blocking buffer. The cells were then washed three times for 5 min and incubated with 50 µl of an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (Invitrogen, cat. no. A21207) diluted 1:500 in blocking buffer for 1 h at room temperature in the dark, after which the cells were washed for 5 min with PBS and stained for 15 min with 50 µl Hoechst (Invitrogen, cat. no. H3570) and HCS CellMask™ Deep Red (Life technologies, cat. no. H32721) diluted 1:10,000 and 1:5000 in PBS, respectively. Finally, the cells were washed twice with PBS and imaged using a Yokogawa CV7000 confocal microscope and the images were analysed using the Columbus Image Analysis software (PerkinElmer).
8
TAK-901 Dose-Response Kinetics Assay
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Cells were seeded into black, clear, flat bottom 96-well plates at 4500 cells per well and allowed to attach overnight at 37 °C, 5% CO2. The next day, serially diluted TAK-901 in DMSO were added to the appropriate wells. DMSO was also added to control wells to a final concentration of 0.1%. At 6 h, 16 h, and 24 h after addition of TAK-901, the cells were washed and fixed with 4% PFA for 20 min at room temperature. The cells were then permeabilised with 0.1% Triton X-100, blocked with 3% BSA, and incubated overnight at 4 °C with p-Histone H3 (S10) antibody (Cell Signalling Technology, cat# 9701, RRID: AB_331535, 1:500). Signals were detected using Alexa Fluor 488 goat anti-rabbit antibody (1:1000) and images were captured using Operetta HCS from Perkin Elmer.
Columbus image analysis software from Perkin Elmer was used to identify and quantify the total p-Histone H3 staining intensity of each well. The positive intensities from the treated wells at each time point were then normalised against the average positive intensity of DMSO treated wells at that time point. The normalised values were plotted against the respective TAK-901 concentrations using GraphPad Prism. Dose response EC50 values were then obtained using 3-parameter non-linear regression.
Columbus image analysis software from Perkin Elmer was used to identify and quantify the total p-Histone H3 staining intensity of each well. The positive intensities from the treated wells at each time point were then normalised against the average positive intensity of DMSO treated wells at that time point. The normalised values were plotted against the respective TAK-901 concentrations using GraphPad Prism. Dose response EC50 values were then obtained using 3-parameter non-linear regression.
9
Immunofluorescence Staining of Fibroblasts
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Fibroblasts were cultured on coverslips or in 384-well plates (BD Falcon) for 24 hours in DMEM. Cells were fixed with 3.7 % formaldehyde at room temperature for 15 minutes, washed twice with PBS, and permeabilized for 15 minutes with 0.1 % Triton X-100 (Sigma) in PBS. Cells were blocked with 3 % bovine serum albumin (Invitrogen) for 30 minutes and then incubated overnight at 4 °C with rabbit anti-TRNT1 primary antibody (Novus Biologicals) diluted 1/50 in PBS buffer containing 2 % BSA and 0.004 % Triton X-100, or mouse anti-TOM20 primary antibody diluted 1/500. Cells were washed and incubated with AlexaFluor-488 anti-rabbit or AlexaFluor-594 anti-mouse antibodies for 1 hour at room temperature. Nuclei were stained with 2 μg/mL Hoechst 33342 (Sigma). Cells were imaged using the Olympus Fluoview FV-1000 Laser Confocal Microscope or using the Opera Imaging system and analyzed using the Columbus Image Analysis Software and Acapella image analysis scripting language (Perkin Elmer).
10
Quantifying Shigella Intercellular Spreading
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For quantification of Shigella spreading, image acquisition was performed using an Operetta (PerkinElmer) automated high-content screening fluorescence microscope at a 10× magnification; a single image was acquired per well, corresponding to an area of ca. 1.4 mm2, approximately 2,000 cells were analyzed per experimental condition and replicate. Image analysis was performed using Columbus image analysis software (PerkinElmer). Briefly, Shigella infection foci were identified based on texture and intensity features, using the ‘Find Texture Regions’ building block implemented in Columbus image analysis software, and the size of each individual foci was determined using the ‘Calculate Morphology Properties’ building block. Infection foci of very small dimension (below 400 μm2; average HeLa-229 area 500 μm2) and containing low bacterial load were excluded since they represent non-productive infections; infection foci touching the borders of the images were also excluded, to improve the overall accuracy of the measurements. At least 500 individual foci from a total of 5 independent experiments were plotted per experimental treatment. Shigella ΔIcsA mutant strain, defective in intercellular spreading was used as a control.
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