Evos fl auto 2 cell imaging system

Manufactured by Thermo Fisher Scientific

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The EVOS FL Auto 2 Cell Imaging System is a fully automated, high-performance microscope designed for live-cell and fixed-sample imaging. It features advanced optics, powerful image acquisition capabilities, and user-friendly software to enable high-quality imaging of a wide range of cell types and samples.

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EVOS FL Auto Imaging System (336 citations) EVOS FL Auto Cell Imaging System (269 citations) EVOS FL Auto 2 Imaging System (161 citations) EVOS FL Auto system (19 citations) EVOS FL Auto Cell Imaging (5 citations) EVOS FL Auto 2 imaging (4 citations) EVOS FL Auto 2 system (4 citations) EVOS FL Auto Cell Imaging System (EVOS FL, (2 citations) FL 2 Auto Imaging System (2 citations) EVOS FL 2 Auto 2 Cell Imaging System (1 citations)

56 protocols using evos fl auto 2 cell imaging system

Cell Viability, Migration, and Invasion Assays

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For the cell viability assay, all cell lines were seeded at a density of 5×10³ in 96-well plates. After 24 hours, cells were serum-starved for 18 hours then continuously exposed to 2% control or 2% DIO mouse serum in SFM for 48 hours. MTT reagent was used to assess cell viability levels as previously described (24), with the absorbance read at 570 nm on a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Instruments Inc., Winooski, VT, USA).
To assess cell migration, all cell lines were seeded in 2-Well Culture-Inserts (ibidi, Munich, GER) in 24-well plates with 7.5×10⁴ cells per insert well. After 24 hours, cells were serum-starved for 18 hours, then inserts removed and cells continuously exposed to 2% control or 2% DIO mouse serum in SFM for 6 hours. The EVOS FL Auto 2 Cell Imaging System (Invitrogen) was used to capture images (x10 magnification) of the wound diameter at baseline and at 6 hours.
To measure invasive capacity, 1.0×10⁴ cells were seeded in SFM in each 24-well Corning Matrigel^® Invasion Chamber (8.0 micron; Corning, NY, USA), and 2% control or 2% DIO mouse serum in SFM was placed beneath the chambers. After 24 hours, the invading cells were fixed in 100% methanol for 1 minute, then stained for 30 minutes with 0.5% crystal violet in 50% methanol. The EVOS FL Auto 2 Cell Imaging System (Invitrogen) was used to capture images (x10 magnification) of the stained cells.

Bowers L.W., Rossi E.L., McDonell S.B., Doerstling S.S., Khatib S.A., Lineberger C.G., Albright J.E., Tang X., deGraffenried L.A, & Hursting S.D. (2018). Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models. Molecular cancer research : MCR, 16(5), 869-879.

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Endothelial Cell Viability Assay

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To exclude potential degeneration of the endothelial cells, we actively tested the endothelial functionality by the ability for the uptake of acetylated low density lipoproteins by the scavenger cell pathway using a commercial assay (Alexa Fluor 488 AcLDL assay, ThermoFisher Scientific, Waltham, MA, USA) [43 (link),44 (link),45 (link)]. For AcLDL assay, the endothelial cells were seeded in 24-well plates and cultivated until 90% confluence. The assay was performed as recommended by the manufacturer. Briefly, the cells were washed two times in a 1% bovine serum albumin (BSA)/PBS⁺ solution for 5 min, followed by adding 400 µL of cell culture medium containing 0.0.125% AcLDL assay solution and incubation at 37 °C for 3.5 h. After adding one drop of NucBlue live staining solution for Deoxyribonucleic acid (DNA) staining, (ThermoFisher Scientific, Waltham, MA, USA), the cells were incubated for another 30 min in the incubator. After washing another three times, images were directly obtained with a conventional fluorescent microscope (EVOS FL Auto 2 cell imaging system, ThermoFisher Scientific, Waltham, MA, USA).

Wacker M., Riedel J., Walles H., Scherner M., Awad G., Varghese S., Schürlein S., Garke B., Veluswamy P., Wippermann J, & Hülsmann J. (2021). Comparative Evaluation on Impacts of Fibronectin, Heparin–Chitosan, and Albumin Coating of Bacterial Nanocellulose Small-Diameter Vascular Grafts on Endothelialization In Vitro. Nanomaterials, 11(8), 1952.

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Astrocyte Activation in Mouse Brain

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The brain tissues of the mice were perfused with a 10% paraformaldehyde solution, followed by a 48 h post-fixation in the same solution. Tissues were then cryoprotected in a 30% sucrose solution for 72 h. Coronal cryosections, 1.0 mm posterior to the bregma and 30 μm thick, were prepared. For the immunohistochemical staining of astrocytic GFAP, brain cryosections were rinsed in Tris-buffered saline (TBS) and treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. After rinsing with TBS and a blocking step with 5% BSA, the sections were incubated at 4 °C overnight with a GFAP-specific antibody (1:300; rabbit polyclonal, Chemicon, Temecula, CA, USA). This was followed by incubation with a secondary antibody conjugated with Alexa Fluor-594 (1:300; Molecular Probes, Eugene, OR, USA). The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) to visualize cellular nuclei. GFAP staining was used in combination with DAPI staining to identify astrocytes based on their characteristic morphology and location within the brain tissue. Following staining, all samples were immediately evaluated and photographed using a fluorescence microscope (EVOS FL Auto2 Cell imaging system; Thermo Fisher Scientific, Waltham, MA, USA). The area of GFAP-positive cells was quantified using ImageJ software.

Yoon E.J., Ahn J.W., Kim H.S., Choi Y., Jeong J., Joo S.S, & Park D. (2023). Improvement of Cognitive Function by Fermented Panax ginseng C.A. Meyer Berries Extracts in an AF64A-Induced Memory Deficit Model. Nutrients, 15(15), 3389.

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Time-lapse Imaging of Cell Cultures

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The EVOS FL Auto 2 Cell Imaging System (ThermoFisher Scientific) was used for imaging cells in culture over time for time-lapse videos. Cells were maintained in a live cell chamber at 37 °C with 5% CO₂ and 85% humidity and areas around neurospheres were scanned every 20 min. Images were compiled at 15 fps using ImageJ to generate time-lapse videos.

Chamling X., Kallman A., Fang W., Berlinicke C.A., Mertz J.L., Devkota P., Pantoja I.E., Smith M.D., Ji Z., Chang C., Kaushik A., Chen L., Whartenby K.A., Calabresi P.A., Mao H.Q., Ji H., Wang T.H, & Zack D.J. (2021). Single-cell transcriptomic reveals molecular diversity and developmental heterogeneity of human stem cell-derived oligodendrocyte lineage cells. Nature Communications, 12, 652.

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3D Spheroid Formation and Cytotoxicity Assay

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Formation of 3D spheroids of HepG2 cells was achieved by seeding cells at a density of 2000 cells/well in an ultra-low attachment 96-well plate (Corning Life Sciences, MA, USA). Once the spheroids formed, they were treated with different concentrations of CP. Media was replaced with a fresh medium containing an appropriate concentration of CP every 48 hr for 12 days. During the incubation, spheroids were observed for their growth in terms of their diameter and surface area using EVOS FL Auto 2 cell imaging system (ThermoFisher scientific, Waltham, USA). On the 12^th day, cells in spheroid were stained and imaged for live/dead cell analysis method using LIVE/DEAD^® Viability/Cytotoxicity kit (Thermo Fisher scientific, Waltham, USA).

Wilcox P.L., Amerson H.V., Kuhlman E.G., Liu B.H., O'Malley D.M, & Sederoff R.R. (1996). Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping.. Proceedings of the National Academy of Sciences of the United States of America, 93(9), 3859-3864.

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Detection of BLV Syncytia Formation

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The blood samples were treated with red blood cell lysis buffer (Abbott Diagnostics Technologies AS, Oslo, Norway) to remove red blood cells, and the pellets were washed with cold phosphate-buffered saline (PBS) and then resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) [53 (link)]. WBCs (1 × 10⁵ cells/well) were applied to the BLV reporter cell lines, CC81-GREMG (5 × 104 cells/well), [23 (link),26 (link),39 (link),54 (link)] that were pre-cultured for one day in a 12-well plate. After 3 days of incubation, the plate was washed with PBS, and fresh DMEM with 10% FBS was added and incubated for up to 48 h. The cells were washed with PBS and fixed with PBS containing 3.7% formaldehyde and 10 mg/mL Hoechst 33,342 (Millipore Sigma). The fixed cells were observed for fluorescent-positive syncytia using an EVOSFL Auto 2 Cell Imaging System (Thermo Fisher Scientific). The permanently BLV-infected FLK-BLV cell line [55 (link),56 (link)] was used as a positive control for this assay.

Bai L., Borjigin L., Sato H., Takeshima S.N., Asaji S., Ishizaki H., Kawashima K., Obuchi Y., Sunaga S., Ando A., Inoko H., Wada S, & Aida Y. (2021). Kinetic Study of BLV Infectivity in BLV Susceptible and Resistant Cattle in Japan from 2017 to 2019. Pathogens, 10(10), 1281.

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Immunofluorescence Staining of 2D Cell Lines

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The 2D cell lines were seeded in a μ-Slide 8-Well chambered coverslip (ibidi, catalog no. 80826) and treated as previously described. Cells were washed with PBS (Gibco) and fixed with 4% paraformaldehyde for 15 min. After a 5-min PBS wash, cells were permeabilized by the addition of PBS containing 0.25% Triton X-100 for 15 min. After two 5-min washes with PBS, cells were incubated with a blocking solution (1% bovine serum albumin in PBS-T (PBS + 0.1% Tween-20)) for 1 h. Cells were then incubated with diluted primary antibody in blocking solution overnight at 4 °C. After three 5-min PBS washes, cells were incubated with diluted secondary antibody in blocking solution for 1 h at room temperature in the dark. After three 5-min PBS washes, coverslips were imaged using an EVOS FL Auto 2 Cell Imaging System (Thermo Fisher Scientific). The antibodies are given in Supplementary Table 6.

Morel K.L., Sheahan A.V., Burkhart D.L., Baca S.C., Boufaied N., Liu Y., Qiu X., Cañadas I., Roehle K., Heckler M., Calagua C., Ye H., Pantelidou C., Galbo P., Panja S., Mitrofanova A., Wilkinson S., Whitlock N.C., Trostel S.Y., Hamid A.A., Kibel A.S., Barbie D.A., Choudhury A.D., Pomerantz M.M., Sweeney C.J., Long H.W., Einstein D.J., Shapiro G.I., Dougan S.K., Sowalsky A.G., He H.H., Freedman M.L., Balk S.P., Loda M., Labbé D.P., Olson B.M, & Ellis L. (2021). EZH2 inhibition activates a dsRNA–STING–interferon stress axis that potentiates response to PD-1 checkpoint blockade in prostate cancer. Nature cancer, 2(4), 444-456.

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Wound Healing and Proliferation Assays

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For wound healing assays, PANC-1 cells were seeded into six-well plates (8 × 10⁵ cells/well), transfected with 50 nM siRNA, and cultured for 24 h, as described above. After treating the cells with mitomycin-C (10 µg/mL) for 2 h, three parallel scratches were made into the confluent monolayer while using a pipette tip. The washed cells were incubated in DMEM without supplements. Images of six randomly chosen areas at the scratches were taken every 3 h for 48 h using EVOS FL Auto 2 Cell Imaging System (ThermoFisher Scientific, Germany). Stacks of all images made for one point within the scratch were generated using Fiji (Version 1.52i) and analyzed by Scratch Assay Analyzer Plugin (MiToBo, Universität Halle) [55 (link)]. Cell migration was calculated in [µm/h]. The velocities were normalized to the control of each cell line. For proliferation assays, the cells were transfected with 33 nM siRNA. 24 h later, cells (5 × 10⁴) were seeded into 12-well plates and then incubated for 12 h to allow for cell attachment. Images of 5–6 randomly chosen areas/well were taken in 12 h interval, using EVOS FL Auto 2 Cell Imaging System. The cells were counted while using Fiji software at time points 0 h (≙ 36 h after transfection) at least 48 h (≙ 84 h after transfection). The doubling time was calculated for each experimental setup.

Meinohl C., Barnard S.J., Fritz-Wolf K., Unger M., Porr A., Heipel M., Wirth S., Madlung J., Nordheim A., Menke A., Becker K, & Giehl K. (2019). Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells. Cancers, 12(1), 30.

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Imaging and Quantification of Resistant Cell Colonies

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Each well was imaged using an EVOS FL Auto 2 Cell Imaging System (Thermo Fisher, MA) at desired time points. The scope and accessories were programmed using the Celleste Imaging Analysis software (Thermo Fisher, MA). Customized MATLAB code was used stitch subplot and generate images of each entire colony. Images were analyzed using Image J (NIH). Outlines of each colony were traced manually in ImageJ. Area measurements were used to calculate colony diameter assuming each colony is a perfect circle. To calculate the fraction of resistant cells in ImageJ, first, the background was removed by setting a pixel threshold. Next, the area occupied by the cells was calculated using the circle and analyze function in ImageJ. As all cells’ nuclei were stained with Hoechst stain (Invitrogen™ NucBlue™ Live ReadyProbes™, ThermoFisher, MA), and all wildtype cells were transduced to express Citrine (25 (link)), the fraction of resistant cells were obtained from the values of (Hoechst – Citrine)/Hoechst. The same background threshold was maintained for all images.

Deb D., Zhu S., LeBlanc M.J, & Danino T. (2022). Assessing chemotherapy dosing strategies in a spatial cell culture model. Frontiers in Oncology, 12, 980770.

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Luminescent Cell Viability Assay for GBM Cells

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GBM cells plated in 96 well plates were transfected with miRNAs as described above. Four days later, each wells were treated with equal volume of CellTiter-Glo Luminescent Cell Viability Assay. After 10 min of incubation, the luminescence intensity was measured by Synergy H1 multi-mode microplate reader (BioTek Instruments, USA). When GBM12-RFP cell was used, the end-point cell viability was imaged by visualizing RFP-positive cells with excitation 532 nm and emission 588 nm using EVOS FL Auto2 Cell Imaging System (Thermo Fisher Scientific, USA).

Yeh M., Wang Y.Y., Yoo J.Y., Oh C., Otani Y., Kang J.M., Park E.S., Kim E., Chung S., Jeon Y.J., Calin G.A., Kaur B., Zhao Z, & Lee T.J. (2021). MicroRNA-138 suppresses glioblastoma proliferation through downregulation of CD44. Scientific Reports, 11, 9219.

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