Cellomics arrayscan

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Cellomics ArrayScan is a high-content screening platform used for automated image acquisition and analysis of cell-based assays. The system captures images of samples and quantifies cellular parameters such as morphology, reporter gene expression, and protein localization.

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Cellomics ArrayScan system ArrayScan VTI Cellomics reader Cellomics ArrayScan VT1 Cellomics ArrayScan™ VT1 HCS automated reader Cellomics ArrayScan VTI analyzer Cellomics ArrayScan XTI Cellomics ArrayScan VTI HCS system Cellomics ArrayScan VTI system Cellomics Arrayscan Platform Cellomics ArrayScan XTI High-Content Screening platform

24 protocols using cellomics arrayscan

Screening Marine Extracts for TAP-1 Upregulation

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To identify marine extracts with potential to upregulate TAP-1 expression in the LMD pTAP-1 cells, the marine extracts were tested at 0.175 mg/mL using the cell-based screening assay. In pre-screenings, the extracts were thawed, rocked for 2 h, and transferred to tissue culture plates with a pin robot. Based on the percentage of activity and cell viability for each extract, the pre-screenings identified seven potentially active extracts. These potentially active extracts were further examined with Cellomics Arrayscan. Extracts dilutions were performed to identify the extract concentration with the highest percent activity. The extracts were stored at −20°C and thawed at room temperature. After thawing, the extract plates were rocked for 2 h. The extracts were serially diluted to 0.5 mg/mL, 0.167 mg/mL, 0.056 mg/mL, 0.0185 mg/mL, 0.0062 mg/mL, 0.0021 mg/mL and 0.00069 mg/mL, and added 24 h after plating the cells in 96-well plates. Following a 48 h incubation at 37°C in 5% CO₂, the cells were fixed as described earlier, Hoechst stained and analyzed with Cellomics Arrayscan.

Nohara L.L., Ellis S.L., Dreier C., Dada S., Saranchova I., Choi K.B., Munro L., Pfeifer C.G., Al Haddad E., Coyle K.M., Morrice J.R., Shim D.J., Ahn P., De Voogd N., Williams D.E., Cheng P., Garrovillas E., Andersen R.J, & Jefferies W.A. (2023). A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours. Frontiers in Pharmacology, 14, 1119607.

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In Vitro Angiogenesis Assay for HUVECs

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At 48 h after siRNA treatment, the HUVECs (4×10⁴ cells/well) in the experimental and control groups were resuspended in endothelial complete medium without serum and seeded onto 96-well plates coated with Matrigel (70 μl). Photomicrographs of the center of each well were obtained following incubation of the cells at 37°C for 48 h. The tubes were stained using a Cellomics Cytoskeletal Rearrangement kit and were analyzed with Cellomics ArrayScan (Cellomics, Pittsburgh, PA, USA). Cell images were acquired with the ArrayScan^® HCS Reader (Cellomics, Pittsburgh, PA, USA). Tube formation was assessed by measuring the tube length by using the Image-Pro Plus 6.0 image processing system (Media Cybernetics, Inc., Rockville, MD, USA). Data are expressed as mm/mm².

YANG W.J., YANG Y.N., CAO J., MAN Z.H., LI Y, & XING Y.Q. (2014). Paxillin regulates vascular endothelial growth factor A-induced in vitro angiogenesis of human umbilical vein endothelial cells. Molecular Medicine Reports, 11(3), 1784-1792.

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High-Throughput Fluorescence Microscopy Imaging

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Images were acquired using a Cellomics Arrayscan (Cellomics Inc.). using a 20 × objective. A minimum of 500 images were acquired per coverslip at 3 channels (green/ red/ blue) per image.

Chao J.T., Roskelley C.D, & Loewen C.J. (2021). MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy. BMC Bioinformatics, 22, 202.

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High-Content Microscopy Image Protocol

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Images were acquired using a Cellomics Arrayscan (Cellomics Inc.). using a 20x objective. 500 images were acquired per coverslip at 3 channels (green/ red/ blue) per image. (1) Images acquired by high-content microscopy are rst pre-processed for quality control (see Figure 3). ( 2)

Chao J.T., Roskelley C.D., & Loewen C. (2020). Functional assessments of PTEN variants using machine-assisted phenotype scoring.

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Quantifying Intracellular Antibody Uptake

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Several antibodies (40-100 µg/antibody) were labeled with a pH-sensitive dye pHrodo Red using a kit from Molecular Probes (cat. no. MP35363). The degrees of labeling (DOLs) for all labeled antibodies were between 1.4 and 2.6. The same endocytosis procedure was used for labeled antibodies, except that Hoechst 33342 was added together with the antibody solution for live cell staining. After endocytosis for 30 min at 37 °C and a brief wash with fresh culture medium, 100 nM LysoTracker Deep Red (Thermo Fisher, cat. no. L12492) was added to stain acidic organelles such as endosomes to confirm the signal specificity of pH-sensitive dye-labeled antibodies. The plates were then analyzed with the Cellomics ArrayScan through three channels of 361, 555, and 647 nm, respectively, for cell nuclei, pH-sensitive labeled antibodies, and intracellular endosomes. The antibody average intensity at 555 nm directly reflects the antibody endocytosis response when it has a good overlay with the signal of endosomes at 647 nm.

Gong S., Li Y., Su W., Ding Y., Lu J., Dong K., Hood S., Zhang W, & Terstappen G.C. (2018). Quantitative Algorithm-Based Paired Imaging Measurement for Antibody-Triggered Endocytosis in Cultured Cells. SLAS discovery : advancing life sciences R & D, 23(8).

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Fixation, Permeabilization, and Imaging of Cells

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After fixation in 4% PFA and transfer to PBS, plates were stored at 4°C for staining. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich T8787) for 15 minutes, washed three times with 1X PBS and stained in a volume of 100 μL with CellMask Deep Red Stain (1:100,000) (ThermoFisher, H32721) for 30 minutes at room temperature in the dark. After staining, cells were washed three times with 1X PBS and stored in 100μL/well 1X PBS. Cells were then imaged using a Cellomics Arrayscan with a 10X objective (ThermoFisher) and analyzed using the HCS Studio quantitative analysis software (ThermoFisher) by defining cellular events based on the non-specific membrane Deep Red CellMask stain in the 650 channel and then quantifying infection by measuring mean fluorescent intensity in the 488nm channel.

Hiatt J., Cavero D.A., McGregor M.J., Zheng W., Budzik J.M., Roth T.L., Haas K.M., Wu D., Rathore U., Meyer-Franke A., Bouzidi M.S., Shifrut E., Lee Y., Kumar V.E., Dang E.V., Gordon D.E., Wojcechowskyj J.A., Hultquist J.F., Fontaine K.A., Pillai S.K., Cox J.S., Ernst J.D., Krogan N.J, & Marson A. (2021). Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins. Cell reports, 35(6), 109105.

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Visualizing Lysosomal Membrane Permeabilization

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Visualization of LMP was undertaken with a method modified from Jäättelä and Nylandsted [46 (link)]. Briefly, A549Pt or A549cisR cells were grown overnight in 96-well plates. Next day, cells were loaded with dextran fluorescein 40 KDa (FITC dextran) for 16 hours. Following a 6 hours chase period, a designated treatment was applied. At the end of experimentation, cells were fixed in ice-cold PBS containing 4% paraformaldehyde for 10 minutes. Cells were then washed with PBS and incubated with Hoechst (2μg/ml) for 30 minutes to stain the nuclei. Plates were mounted on a Cellomics ArrayScan automated fluorescence imager (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were photographed using a 40× objective in 2 fluorescent channels and images of a total of 20 different fields per well were captured. Increased green fluorescence throughout the cytoplasm and loss of the punctae pattern indicate leakage of FITC dextran from the lysosomes, a surrogate of LMP.

Circu M., Cardelli J., Barr M., O’Byrne K., Mills G, & El-Osta H. (2017). Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells. PLoS ONE, 12(9), e0184922.

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Automated High-Content Imaging Analysis

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An automated high content analysis system (Cellomics Array Scan, Thermo Scientific) was used to acquire images by computer-driven (operator independent) collection of 49 valid fields per well with cells in 96 well plates, with >500 cellls (identified by the program as valid primary object) per each sample. Objects were morphometrically and statistically analyzed using the iDev software. Computer-driven identification of primary and secondary objects was based on predetermined parameters, and fluorescent objects (cells, puncta, droplets, total cytoplasm) were quantified using a suite of applicable parameter (including number of objects per cell; total area per cell; total intensity). Nuclei and its associated cytoplasm were sequentially segmented using the Hoechst channel, GFP fluorescent puncta or endogenous LC3 and p62 were revealed by fluorescent antibody staining. Bodipy 493/503, LipidTOX™ Red and LipidTOX™ DeepRed were used to stain lipid droplets. Some studies were carried out using Opera WQEHS and Acapella software (Perkin Elmer; see Supplemental Information). Other methods (time lapse imaging of cultured cells and intravital microscopy are described in Supplementary Information.

Dupont N., Chauhan S., Arko-Mensah J., Castillo E.F., Masedunskas A., Weigert R., Robenek H., Proikas-Cezanne T, & Deretic V. (2014). Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis. Current biology : CB, 24(6), 609-620.

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High-throughput Fluorescent Imaging Analysis

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Bright-field images were captured using an Olympus inverted microscope using cellSens software (Olympus). High-throughput image capture of fluorescent images was acquired at 10× and stitched together using Cellomics Arrayscan (Thermo Fisher Scientific). Image analysis was performed using Cell Profiler (Carpenter et al., 2006 (link)).

Yoon C., Song H., Yin T., Bausch-Fluck D., Frei A.P., Kattman S., Dubois N., Witty A.D., Hewel J.A., Guo H., Emili A., Wollscheid B., Keller G, & Zandstra P.W. (2017). FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors. Stem Cell Reports, 10(1), 87-100.

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Cell Viability Assays for Genotoxic Agents

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293T HEK, MEF and derivative cell lines were cultured in Dulbecco's minimal essential medium (Gibco Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 2 mM l-glutamine. MEF cells were established from day 14.5 embryos and spontaneously immortalized by serial passaging.
For viability assays, 293T HEK or MEF cells were exposed to MMS, MNNG, H₂O₂ or t-buOOH (all chemicals from Sigma, St. Louis, MO, USA) in serum-free medium for 1 h followed by replacement with medium containing serum. UV treatment was performed using a UV cross-linker (Stratagene, San Diego, CA, USA) set at a 254-nm wavelength. HEK 293T and MEF cells were analyzed 24-h post-treatment using either a Muse flow cytometer with the Muse Count and Viability Assay (EMD Millipore, Billerica, MA, USA) or a Coulter counter coupled with Trypan blue staining. For time-lapse video microscopy, cells were plated onto a 96-well plate for 24 h before treatment with 1.0 mM MMS and video capture at 20-min intervals on a Cellomics Array Scan (ThermoFisher Scientific, Waltham, MA, USA).

Jordan J.J., Chhim S., Margulies C.M., Allocca M., Bronson R.T., Klungland A., Samson L.D, & Fu D. (2017). ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage. Cell Death & Disease, 8(7), e2947-.

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