Incucyte zoom instrument

Manufactured by Sartorius

Sourced in United States, Germany

The IncuCyte ZOOM is a live-cell imaging and analysis system designed for real-time, automated, quantitative monitoring of cell-based assays. The instrument enables continuous, noninvasive observation and measurement of cells in their native environment.

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Lab products found in correlation

Well ultra low binding black clear spheroid plates, by Corning (2 mentions) 384 well microplate, by Corning (2 mentions) Opsonizing reagent, by Thermo Fisher Scientific (1 mentions) Cytoone, by USA Scientific (1 mentions) 24 well plate, by Corning (1 mentions) Incucyte cytotox red reagent, by Sartorius (1 mentions) Uvc 500 ultraviolet crosslinker, by Hoefer (1 mentions) Zeocin, by Thermo Fisher Scientific (1 mentions) Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions) Phrodo staphylococcus aureus bioparticles, by Thermo Fisher Scientific (1 mentions) Glucose, by Corning (1 mentions) Gelatin, by Cell Biologics (1 mentions) Incucyte zoom 2018a software, by Sartorius (1 mentions) Penicillin streptomycin p s, by Corning (1 mentions) Dulbecco s modified eagle media, by Corning (1 mentions) Egm 2, by Cell Biologics (1 mentions) 6 well cell culture plate, by Corning (1 mentions) D glucose, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Corning (1 mentions) L glutamine, by Corning (1 mentions)

24 protocols using incucyte zoom instrument

Live Cell Imaging and Apoptosis Monitoring

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The IncuCyte ZOOM instrument (Essen Bioscience) live cell imaging system was used to monitor cell growth. Cells were plated in a 96 Greiner micro clear plate and imaged every 4 hr. The default software parameters for a 96 well plate with a 10x objective were used for imaging. The IncuCyte software was used to calculate mean confluence from two non-overlapping bright phase images of each well.
The IncuCyte ZOOM instrument in combination with the Cell Player 96-well kinetic caspase-3/7 reagent (Essen Bioscience) were used to identify apoptosis by caspase 3/7 activity. The software was used to calculate mean green fluorescence from two non-overlapping fluorescent images of each well. Green fluorescent confluency was normalized to phase contrast confluency to determine apoptosis.

Benedict B., van Harn T., Dekker M., Hermsen S., Kucukosmanoglu A., Pieters W., Delzenne-Goette E., Dorsman J.C., Petermann E., Foijer F, & te Riele H. (2018). Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells. eLife, 7, e37868.

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Real-time Cell Proliferation Assay

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7500 Huh7-LP, 5000 Huh7-HP or 5000 Huh7-Lunet cells per well were seeded in transparent 96-well cell culture plates in at least quadruplicates and four images per well were taken every 2 h with a 10× objective for at least 72 h using an IncuCyte ZOOM instrument (Essen Bioscience, Ann Arbor, MI, USA). Cell growth was quantified using the “confluency mask” of the IncuCyte ZOOM software (version 2015A or newer). Data is given as the mean and standard deviation from four independent wells.

Dächert C., Gladilin E, & Binder M. (2019). Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors. Viruses, 12(1), 36.

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Phagocytosis Assay using pHrodo Bioparticles

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To measure phagocytosis, pHrodo Staphylococcus aureus fluorescent bioparticles (1 mg/ml; ThermoFisher) were resuspended in PBS and vortexed to disperse, and 50 μl of opsonizing reagent (ThermoFisher) was added. The solution was incubated at 37°C for 1 hour. After washing, the pellet was resuspended in PBS. TM cells were plated in 6-well plates (CytoOne; USA Scientific, Ocala, FL, USA) in the presence of shMyo10 silencing lentivirus for 3 days and placed into fresh medium prior to the start of the experiment. For CK-666, TM cells at 70% confluence were pretreated for 2 hours with 100 μM CK-666 in DMSO. Controls included shCtrl silencing lentivirus, 0.04% DMSO vehicle control ,and pretreatment for 30 minutes with 0.78 μM cytochalasin D to disrupt the actin cytoskeleton. Opsonized pHrodo BioParticles (100 μl) were added to each well, and the plate was placed in the Incucyte ZOOM instrument (Essen Bioscience, Ann Arbor, MI, USA). Each well was imaged at 9 points, every 15 minutes for 16 hours using the phase and red fluorescence channels and the 20× objective. At the end of the experiment, the fluorescence intensity at each time point in each well was measured using FIJI. This phagocytosis assay was repeated three times using three biological replicates. Data were combined and averaged and a standard error calculated.

Sun Y.Y., Yang Y.F, & Keller K.E. (2019). Myosin-X Silencing in the Trabecular Meshwork Suggests a Role for Tunneling Nanotubes in Outflow Regulation. Investigative Ophthalmology & Visual Science, 60(2), 843-851.

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Cell Proliferation Assay for EP11313

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In order to monitor cell proliferation in response to treatment with EP11313, cells were seeded in 24-well plates (Corning, NY) at 10⁴ cells per well. Cells were treated with 0, 0.05, 0.1, 0.5, 1, and 1.5 μM EP11313 (n = 4 per treatment) and proliferation was evaluated using the Incucyte Zoom instrument (Essenbioscience, MI).

Copsel S.N., Lightbourn C.O., Barreras H., Lohse I., Wolf D., Bader C.S., Manov J., Kale B.J., Shah D., Brothers S.P., Perez V.L., Komanduri K.V., Wahlestedt C, & Levy R.B. (2019). BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT. Frontiers in Immunology, 9, 3104.

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Real-Time Cell Death and Mitosis Imaging

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A549 NucLight and HeLa NucLight cells were seeded in 96-well plates and incubated at 37° C with 5% CO₂ overnight. The next day, cells were pre-treated with M3541 for 1 hour followed by irradiation (5 Gy). IncuCyte™ Cytotox Red reagent (Essen Bioscience, 4632) was added to the medium to detect dead cells in real-time. Images were taken every 2 h over a 6-day period using an IncuCyte ZOOM instrument (Essen Bioscience). Cell growth curves were plotted as number of fluorescent green nuclei over time. Relative cell death is determined by the counts of IncuCyte™ Cytotox Red labelled cells normalized to total number of green nuclei per well. To monitor mitosis of HeLa GFP-H2B cells they were treated and imaged every 10 min with 20x objective for 7 days. ImageJ (National Institutes of Health) was used to track and analyze the movement of mitotic chromosomes individually.

Chiu L.Y., Sun Q., Zenke F.T., Blaukat A, & Vassilev L.T. (2023). Selective ATM inhibition augments radiation-induced inflammatory signaling and cancer cell death. Aging (Albany NY), 15(2), 492-512.

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Monitoring UVC and Phleomycin-Induced Cell Stress

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HeLa parental and genome-edited cell lines were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum and 2 mM L-glutamine at 37 °C and 5% CO₂. For UVC pulse treatment, cells were seeded in 96-well plates with 100 μL medium, and, after attachment overnight, confluence was monitored at 4 h-intervals in an IncuCyte ZOOM instrument (Essen BioScience) maintained at 37 °C and 5% CO₂. 48 h after seeding, cells were exposed to a UVC pulse (λ = 254 nm, 100 J m⁻²) in a Hoefer UVC 500 Ultraviolet Crosslinker, and confluence was monitored for a total of 160 h. Time points were averaged from eight technical replicates. Control wells on the same plate were exposed to the UVC pulse without removing the plastic cover. For treatment with phleomycin D1, Zeocin (Thermo Fisher Scientific) was added at seeding to a final concentration of 20 μM. When monitoring cell death, SYTOX Green nucleic acid stain (Thermo Fisher Scientific) was added at seeding to a final concentration of 5 μM, and images were taken simultaneously in phase-contrast and green fluorescence modes on an IncuCyte FLR instrument. SYTOX Green nucleic acid stain only penetrates compromised membranes characteristic of dying cells. Green fluorescence was normalized to cell confluence, and the ratio of normalized fluorescence in the absence or in the presence of phleomycin D1 is reported.

Oppikofer M., Sagolla M., Haley B., Zhang H.M., Kummerfeld S.K., Sudhamsu J., Flynn E.M., Bai T., Zhang J., Ciferri C, & Cochran A.G. (2017). Non-canonical reader modules of BAZ1A promote recovery from DNA damage. Nature Communications, 8, 862.

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Quantitative Phagocytosis Assay for Mycobacteria

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Phagocytosis was measured as described previously.18 (link) Briefly, 100-μL opsonized pHrodo Staphylococcus aureus bioparticles (ThermoFisher) were added to each well of a 6-well plate containing GTM or NTM cells. The plate was placed in the Incucyte ZOOM instrument (Essen Bioscience, Ann Arbor, MI, USA), and each well was imaged every 15 minutes for 18 hours by using the phase and red fluorescence channels. Fluorescence at each time point was measured using open-source FIJI software (http://fiji.sc/Fiji). Data are from three technical replicates of >3 GTM and NTM cell strains.

Sun Y.Y., Bradley J.M, & Keller K.E. (2019). Phenotypic and Functional Alterations in Tunneling Nanotubes Formed by Glaucomatous Trabecular Meshwork Cells. Investigative Ophthalmology & Visual Science, 60(14), 4583-4595.

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Pericyte Proliferation Assay

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Pericytes were seeded at 8000 cells/mL into two 6‐well cell culture plate (Corning Inc., Corning, NY, USA) precoated with gelatin (Cell Biologics Inc.) in either Dulbecco's modified Eagle media (DMEM) with 4.5 g·L⁻¹ glucose, l‐glutamine, and sodium pyruvate (Corning Inc.) supplemented with +5% FBS (Corning Inc.) +1% penicillin/streptomycin (P/S) (Corning Inc.) or endothelial growth medium 2 (EGM‐2; Cell Biologics, Inc., Chicago, IL, USA) supplemented with the ECs medium supplement kit (Catalog M1168, Cell Biologics Inc., Chicago, IL, USA). Cells were seeded at 10 000 cells per well into a 24‐well cell culture plate (Corning) precoated with gelatin (Cell Biologics Inc.) in cell culture media. After 6 h for cells to attach, media was changed to include LDL (Sigma) or d‐glucose (Sigma). Plates were placed into an Incucyte Zoom instrument (EssenBioscience Inc., Ann Arbor, MI, USA) integrated with the incucyte zoom 2018A software (EssenBioscience Inc., Ann Arbor, MI, USA), and growth was monitored for 5 days to construct proliferation curves.

Lee L.L., Khakoo A.Y, & Chintalgattu V. (2020). Cardiac pericytes function as key vasoactive cells to regulate homeostasis and disease. FEBS Open Bio, 11(1), 207-225.

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Complement-Dependent Cytotoxicity Assay

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The complement-dependent cytotoxicity assay (CDC) was performed with IgG purified from pre-immunized (day 1) and post-immunized (day 126) sera of cynomolgus monkeys using Zeba™ Spin Desalting Columns (Thermo Scientific) and the Melon Gel IgG Purification Kit (Thermo Scientific). CDC activity was evaluated in Jurkat-GFP cells and MCF-7, SHIN3 and OVCAR cells labeled with 5 μM of CFSE (carboxyfluorescein succinimidyl ester, Life Technologies). Cells (6000/well) were incubated with cynomolgus antibodies at various concentrations and DRAQ7 viability dye (Abcam) at 2 μM, in the presence or absence of 1 % of rabbit complement-MA (Cedarlane) for 2 h in 384-well plates (37 °C). Green fluorescence (live cells) and red (dead cells) fluorescence were measured on an Incucyte™ ZOOM instrument (Essen Bioscience), and the number of cells was calculated using ZOOM software (Essen Bioscience). All measurements were performed in duplicate. The percentage of CDC was determined as follows: % of CDC = ((% of dead cells)^complement – (% of dead cells)^medium) normalized against the control without antibody.

Laubreton D., Bay S., Sedlik C., Artaud C., Ganneau C., Dériaud E., Viel S., Puaux A.L., Amigorena S., Gérard C., Lo-Man R, & Leclerc C. (2016). The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity. Cancer Immunology, Immunotherapy, 65, 315-325.

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Quantifying Drug Response in Cell Growth Assays

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Cells grown for 1 day in full medium were treated with the indicated drugs in 4 different concentrations (0.1, 1, 10 and 100 μM Fig 1B) along with the corresponding DMSO controls on the same plate. The growth of the cells was tracked by phase contrast imaging for 72h with 4 images per well taken every 2h using the Incucyte Zoom instrument (Essen BioScience) and the confluency estimated using the Incucyte Zoom Analysis software (Essen BioScience). The growth rate was estimated with a linear fit on the log-transformed confluency, and the IC50 was determined by fitting a sigmoid of the form:

\begin{matrix} V = \frac{1}{1 + exp (- log (C) + I C 50) \times S} \end{matrix}

to normalised growth rates (implemented in https://github.com/MathurinD/drugResistance). V is the growth rate relative to DMSO control, C is the concentration and the parameters IC50 and slope S are fitted. See S1 Table for the fitted parameters and S2 File for the raw data and analysis scripts, as well as S18 and S19 Figs for example images.

Dorel M., Klinger B., Mari T., Toedling J., Blanc E., Messerschmidt C., Nadler-Holly M., Ziehm M., Sieber A., Hertwig F., Beule D., Eggert A., Schulte J.H., Selbach M, & Blüthgen N. (2021). Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance. PLoS Computational Biology, 17(11), e1009515.

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