Scanr microscope

Manufactured by Olympus

Sourced in Japan, United States

The ScanR microscope is a high-performance digital imaging system designed for automated microscopy and image analysis. It features a modular and flexible architecture that can be configured to meet the specific requirements of various applications.

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Lab products found in correlation

Osteo assay surface multiple well plate, by Corning (2 mentions) On target plus non targeting control sirna 1, by Horizon Discovery (1 mentions) Scanr acquisition software, by Olympus (1 mentions) Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 568 donkey anti mouse, by Thermo Fisher Scientific (1 mentions) Prb 160p, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Sc 22760, by Santa Cruz Biotechnology (1 mentions) Dfc365 fx ccd camera, by Leica camera (1 mentions) C8484 ccd camera, by Hamamatsu Photonics (1 mentions) Dmi6000 b system, by Leica camera (1 mentions) 384 well plate, by Corning (1 mentions) Leica vt1000, by Leica Microsystems (1 mentions) Gelgreen, by Biotium (1 mentions) μclear plate, by Greiner (1 mentions) X tremegene hp dna transfection reagent, by Roche (1 mentions) Scanr analysis software, by Olympus (1 mentions) Facscalibur, by BD (1 mentions) 24 well plate, by Corning (1 mentions) Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)

20 protocols using scanr microscope

Systematic siRNA Screening of Ubiquitin Pathway

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siRNA screening was conducted using the RTF SMARTpool siRNA Library - Human Ubiquitin Conjugation Subset 1 (Dharmacon), consisting of two siRNA-coated plates (H-105615, Lot 12115). Plates were used to reverse-transfect cells at 50 nM final siRNA concentration into optical imaging plates. 72 hr after transfection, eYFP-CRAF degradation was initiated by addition of AUY922 for 8 hr. After incubation, cells were washed and fixed for 10 min using 4% paraformaldehyde in PBS, permeabilized using 0.3% Triton in PBS, and stained with 0.5 μg/mL DAPI in PBS for 15 min. After final wash, all cell samples were immediately imaged using the Olympus ScanR microscope at 10× magnification. Images were subsequently analyzed using ScanR Analysis proprietary software. At minimum 10,000 cells were imaged per individual sample, per experiment.
siRNA verification experiments were conducted in a similar format using specific siRNAs (Table S2). siRNA controls used ON-TARGETplus Non-Targeting Control siRNA #1 (Dharmacon).

Li Z., Zhou L., Prodromou C., Savic V, & Pearl L.H. (2017). HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients. Cell Reports, 19(12), 2515-2528.

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Quantitative Image-Based Cytometry Protocol

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Quantitative image-based cytometry was performed as described previously [67 (link)]. Non-overlapping images were acquired in an unbiased and automated fashion with ScanR acquisition software and the Olympus ScanR microscope. Acquisition time was adjusted for each channel to avoid image saturation and at least 147 images were acquired for each condition. Automated focus was performed using the DAPI channel. Automated image analysis was performed with ScanR image analysis software. ScanR analysis results were exported as .txt files. The .txt dataset was then loaded into R software for further analysis.

Vanzo R., Bartkova J., Merchut-Maya J.M., Hall A., Bouchal J., Dyrskjøt L., Frankel L.B., Gorgoulis V., Maya-Mendoza A., Jäättelä M, & Bartek J. (2019). Autophagy role(s) in response to oncogenes and DNA replication stress. Cell Death and Differentiation, 27(3), 1134-1153.

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Cellular Growth and Chemotherapeutic Assays

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ell images were captured using the Olympus ScanR microscope. The images collected for the dataset were taken in each biological step related to cellular growth and the use of different chemotherapeutic agents and drug schemes. A 20

\times

magnification was used, according to this each image has the dimension of 433

\times

330 m, with a conversion factor 0.32250 m/pixel, and a final pixel per image 16 bit of 1346

\times

1024 pixels. Each brightfield image taken by the microscope was triplicated in the same position by different filters chosen to show the biological structure labeled with the correspondent fluorescence. For the Höechst filter we used an excitation filter of 377/50 with an emission filter of 437–475 nm, for the propidium iodide filter we used an excitation filter of 575/25 with an emission filter of 600–662 nm, and for autophagy and caspase we used an excitation filter of 494/20 with and emission filter of 510–552 nm.

Pattarone G., Acion L., Simian M., Mertelsmann R., Follo M, & Iarussi E. (2021). Learning deep features for dead and living breast cancer cell classification without staining. Scientific Reports, 11, 10304.

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Osteoclastogenesis Assay with RAW 264.7 Cells

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Mouse RAW 264.7 macrophage (pre-osteoclasts) were seeded into 96-well plates at 2 × 10³ cells per well in standard D-MEM for up to 6 days in the presence and absence of RANKL (100 ng/ml) and/or conditioned medium from 5TGM1-GFP MM cells (10% v/v). Mature osteoclasts and their precursors were identified by staining for Tartrate-Resistant Acid Phosphatase (TRAcP). Osteoclast area was visualised by phase contrast microscopy on Olympus ScanR microscope, and area was quantified by Image Analysis using ImageJ.

Marino S., Carrasco G., Li B., Shah K.M., Lath D.L., Sophocleous A., Lawson M.A, & Idris A.I. (2020). JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice. Calcified Tissue International, 107(1), 72-85.

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Immunofluorescence Labeling of Cytoskeletal Proteins

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For immunofluorescence labeling, cells were fixed in ice-cold methanol at −20°C overnight, washed in PBS and permeabilized with TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20). After blocking in 1 mg/ml ovalbumin in PBS for 1 hr at room temperature, the cells were incubated with primary antibody diluted in blocking solution for 1 hr at 37°C. The following primary antibodies were used: rabbit anti-K5 (1:250, Covance, PRB-160P), mouse anti-K8 (1:250, Sigma, #C5301). After washing in PBS, the cells were treated with secondary antibody diluted in blocking solution for 30 min at 37°C. The following secondary antibodies were used: Alexa Fluor® 488 Donkey Anti-Rabbit (1:500, Molecular Probes®, Life Technologies), Alexa Fluor^® 568 Donkey Anti-Mouse (1:500, Molecular Probes^®, Life Technologies). Counterstaining of nuclei was performed by storage in DAPI buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1 M NaCl) containing 0.1 μg/ml DAPI (Sigma).
Subsequently, cells were screened using the Olympus scan^R microscope. Fluorescence intensities and cell sizes were further analyzed using the scan^R analysis software.

Strietz J., Stepputtis S.S., Preca B.T., Vannier C., Kim M.M., Castro D.J., Au Q., Boerries M., Busch H., Aza-Blanc P., Heynen-Genel S., Bronsert P., Kuster B., Stickeler E., Brabletz T., Oshima R.G, & Maurer J. (2016). ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human breast cancer. Oncotarget, 7(50), 83278-83293.

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Automated Copper-Induced Cellular Imaging

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The ChIn_a assay was performed as previously described [7 (link)]. Images were acquired on an inverted automated Olympus Scan⌃R microscope 90 minutes after initial copper treatment. Each well was imaged once per hour for six hours in the channels brightfield, Cy3 and GFP in 4 focal planes (50 μm distance) using a 4x objective (N.A. = 0.13).

Wittmann C., Reischl M., Shah A.H., Kronfuss E., Mikut R., Liebel U, & Grabher C. (2015). A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide. PLoS ONE, 10(10), e0137286.

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Quantifying DNA Damage Response in Ras-Overexpressing Cells

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For deoxynucleosides experiments, overexpression of Ras was induced by incubation with Dox for 8 days. Where indicated, cells were treated with the mix of deoxynucleosides (50 nM of dA, dU, dC and dG) for the whole course of the experiment (Bester et al., 2011) or with 50 μM of cordycepin for the last 2 h of the experiment (Jones et al., 2013). Cells were then fixed with 4% formaldehyde (15 min RT) and permeabilized with 0.1% Triton‐X‐100 (15 min RT). Samples were co‐immunostained with antibodies against 53BP1 (rabbit, sc‐22760, Santa Cruz, 1:1000) and cyclin A (mouse, NCL‐CYCLIN A, Leica‐Novocastra, 1:50), followed by secondary antibodies AlexaFluor568 goat anti‐rabbit and AlexaFluor488 goat anti‐mouse, respectively. To detect γH2AX we used the mouse anti‐γH2AX antibody (613402, Biolegend, 1:500). 250 non‐overlapping images were acquired for each cells‐containing coverslip using the Olympus Scan‐R microscope. At least 4000 cells were scored and images were processed using Scan‐R Analysis software. We scored the total intensity of the pan‐nuclear γH2AX signal in individual nuclei to avoid the variation within an asynchronous cell population (Mistrik et al., 2009; Toledo et al., 2013). For the 53BP1 foci analysis, only cyclin A negative cells were scored. Differences were analyzed using the Wilcoxon signed‐rank test.

Maya-Mendoza A., Ostrakova J., Kosar M., Hall A., Duskova P., Mistrik M., Merchut-Maya J.M., Hodny Z., Bartkova J., Christensen C, & Bartek J. (2014). Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress. Molecular Oncology, 9(3), 601-616.

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Osteoclast Differentiation and Resorption Assay

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Bone marrow (BM) cells were flushed from the long bones of 3-5 week old mice as previously described [53] . M-CSF dependent bone marrow macrophages (osteoclast precursors) were plated into 96-well plates (15 × 10 3 cells/well) in standard alpha-MEM supplemented in the presence of M-CSF (100 ng/ml) for 48 h and then and in M-CSF (25 ng/ ml) and RANKL (100 ng/ml) for up to 5 days. For studies involving prostate cancer cells and their derived factors, M-CSF (25 ng/ml) and RANKL (100 ng/ml) were added 6 h prior to the addition of prostate cancer cells (300 cells/ well) or their conditioned medium (20% v/v) prepared as previously described [53] Cultures were terminated by fixation in 4% paraformaldehyde, and mature osteoclasts were identified by Tartrate-Resistant Acid Phosphatase (TRAcP) staining [53] . Osteoclast activity was assessed by measuring resorbed area in mature osteoclasts cultured on Corn-ing® Osteo Assay Surface multiple well plates (Corning, USA). Resorption pits were visualized on an Olympus ScanR microscope and resorbed area was quantified by using ImageJ software.

Marino S., Bishop R.T., Carrasco G., Logan J.G., Li B, & Idris A.I. (2019). Pharmacological Inhibition of NFκB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo. Calcified tissue international, 105(2).

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Automated Fluorescence Microscopy Protocol

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Cell imaging was performed at room temperature with automated widefield epifluorescence microscopes. With a Scan̂R microscope (Olympus Biosystems)46 (link) equipped with a metal halide light source (MT20), a 12 bit 1344 × 1024 pixel resolution C8484 CCD camera (Hamamatsu) and a 10× UPlanApo objective (Olympus) with a numerical aperture of 0.4. Exposure times at maximum light brightness for Hoechst, mCherry and Alexa Fluor® 647 were 10–20 ms, 450 ms and 850 ms (wt-CFTR) or 15–30 ms, 700 ms and 1000 ms (F508del-CFTR), respectively. The Hoechst channel was used for contrast-based autofocus. Fluorescence images in Fig. 2 were acquired with a Leica DMI6000 B system equipped with a metal halide light source (EL6000) and a DFC365 FX CCD camera (Leica) with a 1392 × 1040 pixel resolution and 12 bit grayscale representation. A 10× HC PL APO objective (Leica) with a numerical aperture of 0.4 was used.

Botelho H.M., Uliyakina I., Awatade N.T., Proença M.C., Tischer C., Sirianant L., Kunzelmann K., Pepperkok R, & Amaral M.D. (2015). Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery. Scientific Reports, 5, 9038.

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High-throughput cell viability assay

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Cells were transferred into 384-well plates (Corning, New York, NY, USA) at a density of 1500 cells/well and incubated overnight at +37°C. The compounds were mixed into medium and pipetted into the wells. Plates were incubated at +37°C for 72 h, and nuclei subsequently stained with Hoechst DNA dye. The number of nuclei was measured with Olympus ScanR microscope (Olympus, Shinjuku, Tokyo, Japan).

Härmä V., Schukov H.P., Happonen A., Ahonen I., Virtanen J., Siitari H., Åkerfelt M., Lötjönen J, & Nees M. (2014). Quantification of Dynamic Morphological Drug Responses in 3D Organotypic Cell Cultures by Automated Image Analysis. PLoS ONE, 9(5), e96426.

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