IncuCyte Zoom: The second generation of long-term dynamic live cell imaging and data analysis systems

IncuCyte Zoom: The second generation of long-term dynamic live cell imaging and data analysis systems
Shanghai Dian Biotech (Shanghai) Ltd.
At present, most of the cell detection methods still use the traditional end point method - just give the final result, and often need to label cells and destroy cells. This method does not allow the true state of the cell to grow, nor does it allow for dynamic monitoring and analysis of the cell's growth process. Essen Corporation of the United States has developed the second generation of long-term real-time dynamic live cell imaging analyzer, IncuCyte Zoom, which uses a non-invasive method to record the real-time growth of cells. This imaging method, called "Live Content Imaging," expands the way users record and understand cell growth, cell behavior, and cell morphology.
IncuCyte Zoom is a live cell imaging analysis platform for non-invasive, long-term real-time dynamics. IncuCyte is divided into two parts: signal acquisition machine and control machine. The signal acquisition machine can be placed in the incubator. There are various sizes of plates, dishes, bottles and slides in the middle. There are photomicrography devices underneath. Micro-photographing, continuous monitoring of cultured cells, remote control, data reading and analysis via a networked computer. The system automatically collects phase contrast images and red/green fluorescence images at each time point. In addition to images or motion recordings in various formats, users can also obtain image-based charts generated by system software based on saturation and count analysis to show changes and trends in cells. For example, a saturation vs. time chart showing cell proliferation, a neurite length vs. time chart showing nerve growth, a relative wound density vs time chart of the Wound Healing experiment, or a vessel length vs time chart showing the neovascularization.
Advantages of IncuCyte: 1) The cultured cells are directly monitored by high-resolution phase contrast microscope or fluorescence microscope. For non-destructive monitoring, the cells can be observed without monitoring, and the image effect is good. 2) The cells do not need to leave the incubator during the monitoring process. No need to worry about the effects of changes in culture conditions on cells; 3) true long-term dynamic live cell imaging, up to days or months, high-resolution phase contrast imaging can eliminate meniscus defects in microplates (meniscus artifacts) ), and without the halo problem of the traditional microscopy system, the cell morphology can be monitored in a 384-well plate; 4) the image processing software automatically quantifies cell proliferation based on saturation and count analysis; 5) automatically outputs cell growth curves and cells Growth video; 6) Real-time monitoring, can change the traditional "end point method" experimental method, rich in data, more accurate and more flexible; 7) high-throughput, can simultaneously monitor 6 standard cell culture plates / dishes / bottles / Slides, including 96-well microplates and 384-well microplates, cells can be tested in 384-well microplates to reduce valuable drug consumption; 8) support More than 200 kinds of standard culture vessels, no special culture vessels are needed, saving the cost of post-experiment; 9) Remote monitoring, data reading and analysis, no need to repeatedly enter and exit the cell room, avoiding potential pollution hazards and manual access problems .
Figure 1: IncuCyte Zoom in the incubator
Compared to the first-generation imager IncuCyte FLR, IncuCyte Zoom is a new design that adds a number of key features, including support for multi-channel fluorescence imaging, support for multiple objective options, increased morphological processing and higher speeds. New features include: 1) IncuCyte Zoom supports high definition (HD) phase contrast, yellow-green fluorescence and red fluorescence auto-imaging, image processing software supports integrated processing of three-channel imaging; 2) users can be in 1 minute 4x, 10x and 20x objectives are replaced by themselves without the help of professionals; 3) because of 12 full-core processors and new hardware and software, the imaging speed is twice that of the first generation FLR; The new camera design makes it more suitable for long-term observation; 5) The new database is designed to make it easier for users to find and retrieve experimental data; 6) Increase the amount of data storage, equipped with 9TB of storage Space and support for external data storage systems; 7) Increased air flow rate, improved heat dissipation and increased scan time; 8) Increased system monitoring, integrated humidity sensor, accelerometer to monitor unacceptable vibration, fan speed and Internal temperature; 9) The user can easily detect and operate the basic status check and operation with the LCD control panel located on the front of the control unit.
Figure 2: IncuCyte internal structure, supporting more than 200 standard culture vessels, can simultaneously observe up to 6 culture vessels
Figure 3: Phase contrast image and proliferation curve of cells recorded by IncuCyte
Figure 3 is a phase contrast image and proliferation curve of cell proliferation recorded by IncuCyte. The left image is a phase contrast image of CHO cells taken at intervals of 12 hours in the same field of view, and the images can be generated continuously for 24 hours without removing the cells from the incubator. The proliferation curve on the right is generated based on an image with an interval of 3 hours. The software automatically converts the image data into quantitative saturation vs. time data to generate a dynamic proliferation curve. The final image will be exported as a separate JPEG file or video.
IncuCyte software features: Users can control the instrument on any networked computer without having to repeatedly take in and out of the specimen and into the cell room, which makes IncuCyte a "virtual incubator". One of the best features of IncuCyte is the intuitive software interface design. The user can define the type of consumables, the time of shooting, the position and the number of fields of view on the software's operator interface. Support for inserting other shooting tasks during long-time shooting to continue shooting to take full advantage of the instrument. IncuCyte autofocuses and automatically collects images. Display control of brightness, size, and contrast can also be performed on the obtained image. Users can customize the sample information, perform group analysis, and electronically mark the container, set keywords, and search the database with the set keywords if necessary, to facilitate the query of images and data obtained at any time in the past. Users can also grab images, charts, and curves directly from the software into Word, Excel, Powerpoint, and Email. All generated data can be archived, and the data and charts can be packaged and sealed for easy retrieval. New features in IncuCyte Zoom include a multi-fluorescence image processing module and a new phase difference imaging processing module. Zoom also designed a new database that makes it easier for users to find and retrieve experimental data.
Figure 4: Software operation interface
IncuCyte application:
(a) Monitoring cell proliferation (Proliferation)
The saturation of the cells can be monitored, or the nuclei can be calibrated with fluorescence to calculate the number of nuclei.
Key features: 1) IncuCyte Zoom combines NucLight Red and NucLight Green reagents to directly monitor cell proliferation; 2) Real-time counting of nuclei under 4×, 10× or 20× objectives; 3) 576 per time point (96-well plate) and 2304 (384-well plate) well data; 4) automatic image acquisition and processing; 5) calculation of doubling time based on nuclear count without lifting cells; 6) growth factors for native The effects of proliferation of primary cells and immortalized cells; 7) The cells did not leave the incubator and all data were image and video verified.
Figure 5: HT-1080 cells transfected with NucLight Red lentivirus reagent were treated with different concentrations of cycloheximide (CHX) and photographed every 3 hours with Zoom under 4x conditions. Although no change in cell morphology was observed, inhibition of cell proliferation based on concentration was observed. The area under the curve (AUC) value was used to calculate the IC50 value of the compound. The doubling time is obtained by calculating the exponential growth formula. The image below is a fluorescent image at 48 hr.
Figure 6: HUVEC cells were transfected with NucLight Red lentivirus reagent, serum starved for 2 hr and cultured in basal medium containing 1% FBS and different concentrations of bFGF (left panel), or bFGF maintained at 10 ng/ml, while Suramin concentration increased. Big (right). Shoot every 3hr with Zoom, the left image is taken with a 4× objective lens, and the right image is taken with a 10× objective lens. Nuclei counts were performed throughout the course of the experiment and EC50 and IC50 values ​​were obtained by calculating the area under the curve (AUC).
(B) Cell Culture QC / Cell Culture Optimization
Monitoring cell morphology changes, quality control of cell culture, such as automated cell culture, using 175T flasks, culture a large number of high-quality cells, can be used as a source of cells for further cell analysis experiments. Monitor changes in growth curve and serum concentration of the medium to find the serum concentration of the most suitable medium. Monitor changes in growth curve and media formulation to find the most suitable media formulation.
Key features: 1) User-defined phase segmentation for high-definition phase contrast imaging; 2) 4×, 10× or 20× images can be collected; 3) Unlabeled phase segmentation indicators use cell saturation to track cells over time Growth status; 4) Ability to detect pro-angiogenic and anti-angiogenic responses; 5) Counting and size observation of clones; 6) Imaging of different parts of the culture flask, Optimize the distribution of cells; 7) Archive images and data for future review of growth characteristics; 8) Optimize growth conditions of native and long-lived cells based on cell saturation; 9) Determine optimal seeding density ; 10) Analyze the microplate layout and promote pipetting technology.
Figure 7: HUVEC cell culture The optimal FBS concentration of HUVEC was > 2.5% in medium containing growth factors and different concentrations of fetal bovine serum (FBS).
Figure 8: HT-1080 cells of different seeding densities were taken every 3 hours for a total of 3 days to obtain an image of real-time dynamic saturation vs. time.
(iii) Monitoring Cytotoxicity
When cytotoxicity occurs, the cell membrane ruptures, and the cytotoxic cells can be stained with the non-permeable dye YOYO-1 and then observed with IncuCyte.
Key features: 1) use NucLight reagent combined with cell non-permeable DNA dye; 2) distinguish between Cytotoxic and Cytostatic; 3) Output data from a two-color analyzer to calculate EC50 and IC50; 4) By obtaining 4×, 10× or 20× high-definition phase contrast images, the cell morphology can be tracked to confirm whether the cells are dead; 5) The process definition can be saved and reused, and YOYO-1 and NucLight can be analyzed for future experiments. Red data.
Figure 9: HT-1080 cells were labeled with NucLight-Red and treated with Camptothecin in the presence of YOYO-1. High definition phase contrast images and fluorescence images are used to confirm cell death.
Figure 10: Calculation of the number of red nuclei and the change of YOYO-1 labeled cells over time using a Basic Analyzer. After data output, the area under the curve (AUC) was used to calculate the IC50 and EC50 values ​​of Camptothecin for HT-1080 cells.
Figure 11: Cytotoxic effects and Cytotostatic Effects of HT-1080 cells induced by Camptothecin (top panel) and Cycloheximide (bottom panel) (bottom panel). The left panel shows the death of cells dynamically displayed by YOYO-1, and the right panel shows the proliferation of cells displayed by NucLight Red.
(4) Monitoring apoptosis (Apoptosis)
The apoptotic agent in the CellPlayer Apoptosis Kit is a linker between the DNA dye and the substrate of the Caspase 3/7 enzyme. If the cell undergoes apoptosis in the Caspase 3/7 pathway, when the reagent is added to the medium, the activated Caspase 3/7 enzyme on the cell membrane degrades the linker, allowing the previously non-luminescent dye to detach from the linker. Binding to the DNA double strand of the cell, causing yellow-green fluorescence, detected by IncuCyte. This allows you to observe the entire process of apoptosis in cells using IncuCyte.
Key features: 1) Label cells with NucLight reagent to detect cell proliferation and dynamically monitor the cytostatic (anti-proliferative) effect of the compound; 2) Add Caspase 3/7 reagent directly to the cell without additional washing steps. To detect apoptosis caused by an intrinsic or extrinsic pathway; 3) to output data from a two-color analyzer to calculate IC50 and EC50. 4) Observe high-resolution phase contrast images with a 4×, 10× or 20× objective to determine Cell death; 5) Quantitative reproducibility: process definitions can be saved and reused to facilitate analysis of Caspase 3/7 and NucLight Red data in future experiments; 96-well and 384-well assays can be performed; Block microplate.
Apoptosis caused by the Intrinsic Pathway:
Figure 12: Cervical adenocarcinoma cells presenting red nuclei were treated with different concentrations of staurosporine (SSP) under conditions of Caspase 3/7 reagent. The process definition accurately measures the number of red nuclei and the number of green Caspase 3/7 objects.
Figure 13: Number of Caspase objects/mm 2 and number of nuclear objects/mm 2 were output by a basic analyzer, and EC50 and IC50 were calculated using the area under the curve (AUC).
Figure 14: Using a basic analyzer, Caspase 3/7 activity (left) and number of nuclei (right) can be detected over time.
Figure 15: Apoptosis caused by extrinsic pathways. A549 lung epithelial cells (Lung Epithelial Cells) were treated with different concentrations of tumor necrosis factor alpha (TNF-α) in the presence of 5 μg/ml cycloheximide, and Caspase 3/7 was quantified using a basic analyzer. active.
(5) Monitoring Cell Migration/Cell Invasion
IncuCyte's Migration module has a 96-well Wound Maker that produces 96 uniform wounds on a 96-well microplate and then records the real-time dynamics of Wound Healing with IncuCyte. .
IncuCyte can get images from a preset point in time and convert them into movies. The system records the initial Wound Mask, and then with the generation of the Revised Wound Mask, the system continuously records images to track wound healing. IncuCyte can also be used for monitoring other cell movements such as "migration" and "invasion". Users can visually observe changes in cell morphology through 2D migration and 3D invasion images, and compare the two cell movement patterns. Based on our high-definition imaging, cells can be viewed without marking. Using a patented algorithm, comprehensive time-lapse-based continuous image acquisition metrics can be obtained, including: relative wound density (patent), wound saturation, and wound width, making the experimental results more quantitative and reproducible. Indicators at all time points can be verified by cell images and movies.
Key features of cell migration/cell invasion: 1) 2D cell migration and 3D cell invasion can be measured by a 4×, 10× or 20× objective in the same 96-well microplate; 2) at each time point Data for 576 wells can be generated (6×96); 3) Non-labeled cell migration/cell invasion can be quantified for both native and long-lived cell lines; 4) Automated analysis can be performed to measure relative wound density, wound saturation, and Wound width; 5) Pharmacological effects can be verified by image and time series recording; 6) Fluorescent cells in cell migration and cell invasion can be photographed and studied using Zoom's two-color fluorescence system.
Figure 16: The left panel is a cell invasion image of HT-1080 cells in 8 mg/ml Matrigel; the right panel is a cell invasion image of MDA-MB-231 cells in 6 mg/ml Matrigel.
Figure 17: The left panel shows the cell migration curves of three cell lines, HT-1080, MDA-MB-231, and MCF-7; the left panel shows the cell invasion curves of two cell lines, HT-1080 and MDA-MB-231. MCF-7 cells did not undergo cell invasion.
Figure 18: Pharmacological study: showing the concentration response of myosin inhibitor (blebbistatin) to HT-1080 cells in cell invasion in 8 mg/ml Matrigel.
(6) Monitoring angiogenesis (Angiogenesis)
Angiogenesis is a complex, multi-step process involving endothelial cell proliferation, endothelial cell migration, and angiogenesis. IncuCyte's CellPlayer Angiogenesis PrimeKit co-cultures GFP-tagged human vascular endothelial cells (HUVECs) with normal human dermal fibroblasts (NHDFs), and co-cultures have neovascularization. Then observe with IncuCyte. IncuCyte Zoom, in conjunction with CellPlayer Angiogenesis PrimeKit, monitors the entire process of angiogenesis and measures the effects of drugs on angiogenesis.
Key features include: 1) use of human primary cells and co-culture systems to show changes in all stages of the angiogenesis process; 2) two options for living cells and frozen cell kits; 3) combination of Zoom and angiogenesis software Automatically acquire time-series images, providing dynamic indicators such as vessel length, vessel area, and number of branch nodes; 4) Dynamic readings can study complex pathways including VEGF (vascular endothelial growth factor) and non-VEGF (non-vascular endothelial growth factor) Signal-mediated angiogenesis and vascular breakdown; 5) ability to measure pro-angiogenic and anti-angiogenic effects; 6) all data are verified by images and films; 7) The reaction was carried out in a 96-well microplate, and the data was quantitative and repeatable.
Essen offers the CellPlayer Angiogenesis Prime Kit-Cryo, the CellPlayer Angiogenesis Prime Kit-Live, and the angiogenesis outsourcing service. Specific kit specifications and experimental outsourcing services can be found on the Essen website.
Figure 19: Angiogenesis, on day 1 and day 14, the vessel length increased from 0.6 mm/mm 2 to 11.6 mm/mm 2
Figure 20: Dynamic effects of different concentrations of vascular endothelial growth factor (VEGF) on angiogenesis.
Another kit is CellPlayer Angiogenesis StemKit, which co-cultures GFP-labeled endothelial colony-forming cells (EDFC) with adipose-derived stem cells (ADSC). There will be neovascularization and then observed with IncuCyte. IncuCyte Zoom, in conjunction with CellPlayer Angiogenesis StemKit, monitors the entire process of angiogenesis and measures the effects of drugs on angiogenesis.
Key features include: 1) a co-culture stem cell model of human origin; 2) provided as a cryo-kit; 3) faster angiogenesis than the PrimeKit kit; 4) Pericyte biology; 5) Dynamic reading of angiogenesis and vascular decomposition; 6) All indicators have images and video for verification; 7) Experiments can be performed on 96-well microplates with good repeatability (Z'>0.8).
Essen offers the CellPlayer Angiogenesis StemKit in frozen form and the angiogenesis laboratory outsourcing service. Specific kit specifications and experimental outsourcing services can be found on the Essen website.
Figure 21: Angioblastin (angiopoietin 2, Ang-2) was added to the established vascular network and dynamic data showed angiogenic breakdown.
(VII) Monitoring Report Gene (Reporter Gene)
The cells were transfected with a GFP-containing vector, and the promoter to be studied was inserted upstream of GFP. This allows the activity of the promoter or the expression activity of the reporter gene to be monitored by IncuCyte to observe the fluorescence intensity of GFP and the number of cells that fluoresce.
In contrast to the traditional endpoint luciferase method, the key features of IncuCyte are: 1) Data rich: 96-well real-time dynamic data can obtain insights that are not available in the endpoint method; 2) Cost savings: no lysis required, no luciferase method required Requires terminal reaction substrate, saving time and cost; 3) Convenience: Real-time dynamic reading allows the user to optimize the signal window in a single experiment without prior decision on when to terminate the experiment; 4) Sensitivity: available under each condition Multiple time point data increases the quantitative and robustness of the experiment; 5) Customizable: The user can customize the promoter as needed, modify the reaction system, and monitor the effect of the drug on the reporter gene.
Essen can provide outsourcing services for this experiment according to customer needs. For details of outsourcing services, please refer to the Essen website.
Figure 22: Activity of NF-κB expressed by rhGFP reporter gene in HEK293 cells induced by rhTNF-α stimulation. In HEK293 cells transfected with the pNF-κB-rhGFP reporter gene, treatment with 3-fold dilution of rhTNF-α was performed. Images were taken at intervals of 15 min.
The image indicates that the higher the concentration of the external rhTNF-α, the greater the fluorescence saturation of the cells, indicating that the activity of NF-κB inside the cells is stronger. The subject fluorescence saturation indicates the expression activity of the reporter gene and/or the activity of the promoter.
(VII) Nerve Growth Tracking (Kinetic NeuroTrack)
IncuCyte Zoom quantitatively analyzes the growth of neurons in neurites, resulting in images and growth curves.
Key features include: 1) Non-labeled real-time dynamic quantitative analysis of neurite dynamic growth (eg initial state, branching, extension, retraction); 2) automated image acquisition and integrated analysis software for accurate neurological optimization Outburst detection and data analysis; 3) Multiple experimental capabilities, parallel monitoring of high-definition phase contrast imaging and multi-channel fluorescence imaging with IncuCyte Zoom; 4) Compatible with a variety of neuronal cell types including cell lines and blasts.
Figure 23: Neurite growth of murine E18 cortical neurons in 96-well microplates. A) 20-fold phase contrast images of primitive neurons after 96 hours of inoculation; B) image segmentation of neurites (yellow) and cell population (purple); C) neurites at different concentrations of protein kinase C inhibitor Ro Inhibition over time by -31-8220 (mean±SD, n=6 per condition)
Figure 24: Neurite growth of iPSC-derived iCell neurons in 96-well microplates. Left panel: Growth of neurites over time in iCell neurons at different seeding densities (mean ± SD, n=6 per condition). Right panel: 20-fold phase contrast image of iCell neuron cells inoculated at 10 k cells/well at 96 hours.
Figure 25: Neurite growth of neuronal-2a NucLight Red cells in 96-well microplates. Left panel: 20-fold mixed image of phase contrast imaging and red fluorescence imaging after 78 hours of treatment with 16 μM all-trans retinoic acid. Right panel: Length of neurites that changed with increasing concentration of all-trans retinoic acid after 78 hours of treatment, which has been normalized to the number of cells (mean ± SD, n = 10).
IncuCyte Zoom application summary:
1) Monitoring cell proliferation (Proliferation): non-invasive, quantitative analysis of cell proliferation by real-time dynamic imaging;
2) Cell Culture QC/Cell Culture Optimization: Dynamic growth curve based on cell saturation can be generated by real-time dynamic imaging;
3) Monitoring Cytotoxicity: Mixing the dye and cells and then reading, the operator can leave, is a dye-based experiment;
4) Monitoring Apoptosis: mixing the apoptotic reagent with the cells and then reading, the operator can leave, is a dye-based experiment;
5) Cell Migration/Cell Invasion: A highly consistent 96-well wound scratching tool that dynamically monitors the wound healing process in real time to obtain quantitative indicators of cell movement;
6) Monitoring Angiogenesis: Using a validated angiogenesis kit with quantitative indicators for dynamic analysis of angiogenesis;
7) Reporter Gene: Real-time dynamic data can be obtained without lysis of cells;
8) Kinetic NeuroTrack: Quantitative analysis of the growth of neurites in neuronal cells.
to sum up:
Some of the key, important application notes above demonstrate the role of the IncuCyte Zoom system in quantitative, non-invasive, real-time cell analysis. Unmarked, non-invasive imaging techniques and data analysis allow users to access high-quality images, videos and data. Unlike other cells that use electronic impedance to monitor cells, IncuCyte can record cell morphology, get images and dynamic video. In addition, Essen has developed more live cell kinetic assays and reagents, which are used in conjunction with Zoom for assay development, technology assessment, and outsourcing services. For-service outsourcing) and drug discovery partnerships.
Ordering Information: IncuCyte Zoom is available in HD and 2CLR . HD can only be used for phase contrast imaging, while 2CLR can be used for phase contrast and red / green two-color fluorescence imaging, and HD can be upgraded to 2CLR . The kit includes: a cell migration / invasive kit (including analysis software, 96- well scarer Wound Maker and 15 96- well ImageLock microplates), a neural tracking kit (including analysis software), and an angiogenesis kit (including analysis software).
For more information, please refer to the Essen website: and the Codex website:
Shanghai Dian Biotech Co., Ltd. Contact: Room 2101, Building 6, Huiyang Building, 1139 Pudong Avenue, Shanghai. Tel: (+86) 21-58605185, fax: (+86) 21-51973282, E-mail:

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