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Characteristics and application of IncuCyte, a long-term dynamic living cell imaging and data analysis system
IncuCyte: Unlabeled, long-term dynamic live cell imaging analyzer
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 an unlabeled, long-term dynamic live cell imaging analyzer, IncuCyte, which uses a non-invasive method to record the real-time growth of cells.
IncuCyte is a long-lived, non-invasive live cell imaging analysis platform. 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 by connecting computers and networks. The system automatically aggregates images at each point in time and automatically generates dynamic recordings. 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. IncuCyte FLR captures phase contrast images and yellow-green fluorescence images showing GFP, YFP, Fluorescein, Alexa 488, nuclear dyes, etc. IncuCyte EX can be integrated with automated flat and liquid handling systems. They are optimized for optical components, with clear imaging and no traditional halo problems.
Figure 1: IncuCyte in the incubator
Advantages of IncuCyte: 1) The cultured cells are directly monitored by high-definition (HD) phase contrast microscope or fluorescence microscope. For non-destructive monitoring, cells can be observed and monitored without staining, and the image effect is good; 2) cells are not needed during monitoring Leave the incubator without worrying about the effects of changes in culture conditions on the cells; 3) True long-term dynamic live cell imaging for days or months, high definition (HD) phase contrast imaging can eliminate microplate bends Meniscus artifacts, without the halo problem of traditional microscopy systems, can monitor cell morphology in 384-well microplates; 4) image processing software automatically quantifies cell proliferation based on saturation and count analysis; 5) Automated Output cell growth curve and cell 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 cultures Plate/dish/bottle, cells in a 384-well plate can reduce valuable drug consumption; 8) Support more than 200 standard culture vessels, no
Figure 2: IncuCyte internal structure supporting more than 200 standard 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: With networked software, 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, making IncuCyte a “virtual incubatorâ€. Users can define the type of consumables, the time and location of the shooting on the software's operator interface, and support the insertion of 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.
Figure 4: Software operation interface
IncuCyte application:
(a) Monitoring cell proliferation (Proliferation)
The morphological changes of the cells can be monitored, and the nuclei can be calibrated by fluorescence to observe the size and count of the nuclei.
Key features: 1) quantitative dynamic process indicators obtained from real-time images; 2) non-invasive indicators such as single-layer saturation; 3) labeled indicators (GFP or live cell staining) for nuclear count; 4) all dynamics The signal has image and video verification.
Figure 5: Growth curves of different cells, photographed every 3 hours, automatically generate single layer saturation VS. time chart
(B) Cell Culture QC and 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) Obtain real-time images of proliferating cells without labeling and moving cells out of the incubator; 2) Optimize the culture process with dynamic indicators such as monolayer saturation; 3) Excellent quality control of the microplates, The patented HD (high definition) imaging mode eliminates meniscus artifacts from microplates; 4) evaluates cell growth with object algorithms and eliminates human interference; 5) the system can observe large areas of cell culture vessels, Increased the accuracy of the assessment; 6) All indicators have images and videos for verification.
Figure 6: Proliferation curve of CHO-hERG cells in different serum concentration media, monolayer saturation vs. time chart, cell growth in 2% serum medium faster than growth in 10% serum medium
(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) Dynamic reading: It can record the short-term and long-term changes of cell activity in different physiological environments in real time. It is not necessary to determine a widely applicable endpoint in advance; 2) Mix and then image: Add YOYO-1 directly In the cells of the culture medium, the fluid aspiration steps are removed, and the cell disruption of the damaged cells is eliminated; 3) automatic data acquisition: phase difference images and fluorescence can be automatically collected in the incubator Image; 4) Cell morphology distinction: High contrast phase contrast images and image data allow the user to qualitatively distinguish the different effects of Cytotoxic and Cytostatic (anti-proliferation); 5) High reproducibility: Real-time dynamic cell death records are highly reproducible Z' is between 0.6 and 0.8.
Figure 7: Toxicological experiments of different concentrations of Taxol on MDA-MB-231 cells. Taxol was added at 24 hours and 48 hours, photographed every 3 hours, and automatically generated monolayer saturation vs. time chart
(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) Dynamic and continuous observation of apoptosis; 2) Unique “no-clean†labeling procedure to make quantification more convenient and avoid washing away dead or dying cells during labeling; 3) and flow Different cytometers, cells can be analyzed without moving, the same cells can be used for subsequent dynamic studies; 4) dynamic process indicators can be obtained from real-time images, including apoptotic target counts or apoptosis Standardization of the target area; 5) phase contrast images can be used to monitor changes in cell morphology associated with apoptosis; 6) after the end of the experiment, the total number of DNA is measured by dye staining to obtain an apoptotic index indicating apoptosis. Percentage of objects; 7) Can be used for quantitative analysis of 96-well microplates.
Figure 8: Fluorescent image of apoptosis
Figure 9: Serum starvation-induced apoptosis in HUVEC cells. After addition of Caspase 3/7 apoptosis reagent, apoptotic cells can fluoresce and are detected by IncuCyte FLR. The activity of Caspase 3/7 can be quantified using a target count or target saturation algorithm.
(5) Monitoring Cell Migration
(vi) Monitoring Cell Invasion
Figure 10: Wound healing, cell migration and cell invasion
IncuCyte's Migration module features 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 proprietary algorithm, a comprehensive time-lapse-based continuous image acquisition indicator can be obtained, including: relative wound density (patent), wound width, and wound saturation, which makes 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: 1) no labeling required; 2) 96-well quantitative dynamic data suitable for path analysis, optimized dynamic range, Z'>0.7; 3) 2D migration compatible with ECM (extracellular matrix) coating ; 3) Dynamic process indicators can be obtained from real-time images, including wound relative density (patent), wound width, and wound saturation. 4) All indicators have image and video verification.
Key features of cell invasion: 1) Reproducible pharmacology: This experiment allows the user to assess the pharmacological effects of different reagents on the cells in the presence of extracellular matrix (ECM); 2) Cell morphology: can be pre-established The way to obtain images allows the user to observe the different morphological changes of the cells during 2D migration and 3D invasion; 3) Contrast: Users can set different ECM concentrations on the same microplate, compare migration and invasion; 4 No need to mark: HD imaging technology does not require cell labeling; 5) Dynamic reading: Users can simultaneously observe the speed and extent of invasion under different experimental variables, can be used to explore time-based pharmacology, to optimize experiments (such as ECM concentration, cells Density), increased experimental sensitivity; 6) Quantitative: When using the patented Wound Relative Density (RWD) indicator, the results are quantitative and repeatable.
Figure 11: Inhibition of migration of HT 1080 cells by Cytochalasin D, relative wound density (RWD%) vs. time chart, the greater the concentration of Cyto D, the slower wound healing
Figure 12: Comparison of cell migration (odd column) and cell invasion (even column), wound relative density (RWD%) vs. time chart
(7) Monitoring angiogenesis (Angiogenesis)
Angiogenesis is a complex, multi-step process involving endothelial cell proliferation, endothelial cell migration, and angiogenesis. IncuCyte's GFP-AngioKit transfects human umbilical cord endothelial cells (HUVECs) with GFP-lentivirus and then co-cultures with normal human dermal fibroblasts (NHDFs). The co-cultures have neovascularization and then IncuCyte observes. IncuCyte FLR and GFP-AngioKit can be used to monitor the entire process of angiogenesis and determine the effect of drugs on angiogenesis.
Key features include: 1) the use of human primary cells, and co-culture systems to show changes in all stages of the angiogenesis process; 2) the experiment is very accurate, Z'>0.7; 3) dynamic readings can study complex pathways, including VEGF (vascular endothelial growth factor) and non-VEGF (non-vascular endothelial growth factor) signaling-mediated angiogenesis; 4) time-series readings can study the effects of drugs on time-based effects, such as vascular mitigation; 5) measurable Pro-angiogenic and anti-angiogenic anti-angiogenic effects; 6) Quantitative indicators derived from real-time image acquisition, including: vessel length, vessel area, and branch points; 7) All data can be verified with images and films.
Figure 13: Angiogenesis, on day 1 and day 14, the vessel length increased from 0.6 mm/mm 2 to 11.6 mm/mm 2
Figure 14: Inhibition of angiogenesis by Suramin in the presence of VEGF. The greater the concentration of Suramin, the smaller the increase in vessel length and the more pronounced inhibition.
(8) 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 end-point luciferase method, the key features of IncuCyte are: 1) data rich: real-time dynamic images and data at multiple time points not available at the end point method; 2) cost savings: no need for lysis, no fluorescence The enzymatic method requires a terminal reaction substrate, saving time and cost; 3) Convenience: Dynamic data allows the user to optimize the signal window in a single experiment without prior decision on when to terminate the experiment; 4) Sensitive: available for each Multiple time point data under conditions increase the quantitative and robustness of the experiment; 5) Customizable: the user can customize the promoter according to needs, modify the reaction system, and monitor the effect of the drug on the reporter gene.
Figure 15: 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. Object Confluence indicates the expression of a reporter gene and/or the activity of a promoter.
(9) Cell Differentiation: Long-term dynamic analysis of cells in an incubator.
IncuCyte FLR application summary:
1) Monitoring of cell proliferation (Proliferation): non-invasive, quantitative analysis of cell proliferation by real-time dynamic imaging; 2) Cell Culture QC/Cell Culture Optimization: by real-time Dynamic imaging, which generates dynamic growth curves based on cell morphology and saturation; 3) monitoring of cytotoxicity: mixing dyes and cells and readings, the operator can leave, is a dye-based experiment; 4) monitoring apoptosis (Apoptosis): Mixing apoptotic reagents with cells and reading them, the operator can leave, is a dye-based experiment; 5) Monitoring Cell Migration: a highly consistent 96-well wound scratch tool for real-time dynamic monitoring The wound healing process can obtain quantitative indicators of cell movement; 6) Cell Invasion: A 96-well wound scratching tool with high consistency, real-time dynamic monitoring of wound healing process, quantitative indicators of cell movement 7) Monitoring Angiogenesis: Using validated blood vessels Health kit, with quantitative analysis of dynamic analysis of angiogenesis; 8) Reporter Gene: Real-time dynamic data can be obtained without lysis of cells; 9) Cell Differentiation: Cells are cultured in an incubator Long-term dynamic analysis and so on.
to sum up:
Some of the key, important application notes above demonstrate the role of the IncuCyte 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. 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: