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Clear, detailed publication-quality images and quantitative data are what the CellInsight High-Content Screening platforms are engineered for. Review sample applications, including fluorescence images, graphs, and videos for life sciences applications such as angiogenesis, apoptosis, autophagy, cell cycle and proliferation, endocytosis, and viability. Browse our antibodies and assays. Do not forget to check out our image gallery below.
The Thermo Scientific CellInsight high-content platform allows you to take advantage of the entire fluorescent spectrum to optimize your assay—and multiplex your components to ask more in-depth biological questions. To that end, we have a myriad of antibodies and assays compatible with the CellInsight systems. Below is a comprehensive list of applications using assays together with the high-content platform.
This reliable, automated assay identifies angiogenic tubes (micro-capillaries) formed by endothelial cells and provides quantitative measurements related to angiogenic tube formation such as the number of tubes, their morphology and branching, and an angiogenic index.
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HUVEC cells, grown in Matrigel support and treated with angiogenic compounds and imaged at 5x magnification.
(Left) Cell nuclei are stained with Invitrogen Molecular Probes Hoechst 33342 (shown in red) and the cytoskeleton (actin fibers) is stained using a phalloidin conjugate (shown in green/yellow; wide choice of labels). (Right) Angiogenesis features identified using Thermo Scientific HCS Studio Software. In a two-channel assay, connected tubes are automatically identified using a blue overlay, and branching nodes are displayed as pink spots. Multiple parameters can be automatically measured in additional detection channels and correlated with the identified tube structures.
Caspase assay for HCS that allows the simple detection of active caspases in living cells in real time or in fixed cells.
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This assay uses a nuclear stain to identify cells and a TUNEL label to measure DNA strand breaks. The Invitrogen Click-iT TUNEL Alexa Fluor Imaging Assay kits are fast and efficient and offer precise, quantitative data even with high levels of apoptotic cells. The kits are optimized for HCS and provide a choice of three wavelength options to aid in multiplexing with other cellular measurements.
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In this assay, quantitation of LC3B protein on autophagic vesicles is achieved using a fixed end-point assay based on immunofluorescence detection. Cells are identified using a nuclear stain and LC3B expression associated with each cell is measured.
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A549 cells treated to induce autophagy and imaged.(Left) A549 cells were treated with chloroquine and stained with Invitrogen Hoechst 33342, Invitrogen HCS CellMask Deep Red, and an anti-LC3B with Invitrogen Alexa Fluor 488 goat anti-rabbit antibody. Cells accumulate LC3B at higher chloroquine concentrations. (Right) Using Thermo Scientific CellInsight CX5 high-content platform and Thermo Scientific HCS Studio Software, autophagy features were automatically identified: nuclei (blue), cells (green boundary), and LC3B was assayed both by granule count (pink) and by measuring fluorescence intensity in the 488 channel.
Cell cycle assays and mitotic index measurements provide insight into cell division and quantify the effects of compounds or treatments that impact mitotic progression. Automated assays can measure cellular DNA content as an indicator of where in the cycle a cell was stained, or you can detect the levels of proteins associated with mitosis such as Histone H3.
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This automated assay measures new DNA synthesis based on incorporation of the nucleoside analog EdU into DNA. A copper-catalyzed “click” reaction adds a fluorescent Invitrogen Alexa Fluor dye conjugate to the EdU and enables detection with a range of wavelength choices for easy multiplexing.
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This assay automatically measures changes in cell morphology reflecting changes in the underlying intracellular structure. Specifically, the assay measures the morphology of the whole cell and the nucleus, as well as the number, dimensions, intracellular location, and arrangement of F-actin and microtubule fibers.
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Cytoskeletal rearrangement assay. A549 cells were treated with cytochalasin-D and analyzed using the Thermo Scientific CellInsight CX7 High-Content Analysis Platform. Nuclei are labeled with Invitrogen HCS NuclearMask Blue Stain and F-actin is labeled with Invitrogen Alexa Fluor 488 phalloidin, and the whole cell is demarcated using Invitrogen HCS CellMask Deep Red Stain. The assay automatically segments the cells (yellow overlay) and identifies the location and orientation of actin fibers (green overlay). Actin fibers greater than a threshold length are identified and labeled (red overlay). Individual cells can be identified from bar charts and scatter plots.
The HCS DNA Damage Kit uses a secondary antibody conjugate to detect phosphorylated H2AX, Image-iT DEAD Green dye to detect cytotoxicity, and Hoechst 33342 to label nuclei in both live and dead cells.
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Endocytosis is involved in many cellular processes such as nutrient acquisition and receptor signaling. Analysis of the extent of internalization of a given molecule can be used as a marker of endocytosis. Commonly used markers include ligands such as LDL, EGF, or transferrin as well as fluid phase markers, for example 10,000 MW dextrans conjugated to fluorophores to determine their localization.
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U2OS cells stably expressing a GFP-EGF receptor construct and incubated with pHrodo red EGF conjugate were treated with PitStop 2 inhibitor (Abcam, Inc.) to inhibit endocytosis. Cells were stained with Invitrogen Hoechst 33342, and pHrodo red EGF conjugate. Stained cells were imaged using Thermo Scientific CellInsight CX5 high-content platform (row A) and analyzed using HCS Studio Software. Internalized EGF receptors and the EGF ligand were automatically identified (row B): nuclei (blue), cells (green boundary), and EGF or the EGF receptor were assayed both by granule count (red for pHrodo EGF or green for GFP-EGF receptor).
HCS LipidTOX neutral lipid stains were developed for image-based high-content screening (HCS) assays to characterize the potentially toxic side effects of compounds on lipid metabolism in mammalian cell lines. The LipidTOX assay series includes neutral lipid stains for fixed cells and phospholipid stains for live cells, which can be combined in multiplexed assays.
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HCS LipidTOX Deep Red Neutral Lipid Stain, for cellular imaging | HCS LipidTOX Red Neutral Lipid Stain, for cellular imaging | HCS LipidTOX Green Neutral Lipid Stain, for cellular imaging | |
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Readout | High affinity for neutral lipid droplets in fixed cells | ||
Common filter set | Cy5 | Texas Red | FITC |
Reporter | LipidTOX Deep Red neutral lipid stain | LipidTOX Red neutral lipid stain | LipidTOX Green neutral lipid stain |
Ex/Em (nm) | 637/655 | 577/609 | 495/505 |
Live-cell compatible | No | ||
Added to growth media | No | ||
Formaldehyde fixable | Cells labeled after fixation | ||
Multiplexing | Can be multiplexed with phospholipidosis detection reagents (see tables below) | ||
Platform | Imaging, HCS | ||
Bibliography | Citations | ||
Format | 125 μL (1,200 assays for steatosis/240 assays for adipogenesis) | ||
Cat. No. | H34477 | H34476 | H34475 |
HCS LipidTOX Phospholipidosis and Steatosis Detection Kit | HCS LipidTOX Phospholipidosis and Steatosis Detection Kit | |
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Readout | Kit for the sequential analysis of phospholipidosis and steatosis | |
Common filter set | Texas Red | FITC |
Reporter | LipidTOX Red phospholipid stain | LipidTOX Green neutral lipid stain |
Ex/Em (nm) | 595/615 | 495/505 |
Live-cell compatible | Yes | No |
Added to growth media | Yes | No |
Formaldehyde fixable | Yes | Labeled after fixation |
Multiplexing | Combination assay kit for HCS | |
Bibliography | Citations | |
Platform | Imaging, HCS | |
Format | 2 plates/240 assays | 10 plates/1,200 assays |
Cat. No. | H34157 | H34158 |
HCS LipidTOX Green Phospholipidosis Detection Reagent | HCS LipidTOX Red Phospholipidosis Detection Reagent | |
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Readout | Labels phospholipid accumulation after incubation with live cells | |
Common filter set | FITC | Texas Red |
Reporter | LipidTOX Green phospholipid stain | LipidTOX Red phospholipid stain |
Ex/Em (nm) | 495/525 | 595/615 |
Live-cell compatible | Yes | |
Added to growth media | Yes | |
Formaldehyde fixable | Yes | |
Multiplexing | Can be multiplexed with neutral lipid stains (see table above) | |
Bibliography | Citations | |
Platform | Imaging, HCS | |
Format | 125 μL (1,200 assays) | |
Cat. No. | H34350 | H34351 |
This automated assay uses Invitrogen MitoTracker Orange as an indicator of mitochondrial function because its accumulation in the mitochondria of live cells is proportional to the mitochondrial membrane potential. Invitrogen Hoechst 33342 is used as a segmentation tool to identify cells; a viability stain, such as the Invitrogen Image-iT DEAD Green Viability Stain, is easily incorporated to further multiplex the assay. These three dyes have sufficient retention of fluorescence signal intensity upon formaldehyde fixation and detergent permeabilization to be useful in fixed endpoint assays, as well as applications involving immunocytochemistry for specific protein detection.
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This automated assay enables quantitation and correlation of neuron and neurite morphology. Analysis can range from simple neurite outgrowth measurement to complex analysis of extended neurite outgrowth and branching.
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Using fluorogenic reporters such as CellROX dyes allows for the real-time analysis of ROS generation in living cells and in fixed-cell preparations.
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The Click-iT Plus OPP Alexa Fluor Protein Synthesis Assay Kit provides a fast, sensitive method for the detection of protein synthesis in a HCS format. The assay incorporates O-propargyl-puromycin (OPP) efficiently into newly translated proteins in a complete methionine-containing medium. The protein is then fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly specific, mild click reaction.
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Hap1 cells treated with cyclohexamide and labeled with Hoechst 33342 and Click-iT OPP followed by addition of an Alexa Fluor 488 azide for chemoselective detection (Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit). The cells were imaged using a Thermo Scientific CellInsight CX5 high-content platform CellInsight CX5 high-content platform (row A) and Thermo Scientific HCS Studio Software, which were used to automatically identify (row B) and quantify the intensity of Click-iT OPP green fluorescence from each cell.
This automated assay monitors the transition of pluripotent stem cells (PSC) to a mesodermal and then committed cardiomyocyte phenotype using a fixed-cell imaging protocol. In a 3-channel assay, cells are fixed and stained with a nuclear stain for localization and with primary antibodies against nuclear transcription factors associated with pluripotency (Oct4) and cardiac differentiation (Nkx2.5).
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This automated assay uses the co-localization of presynaptic and postsynaptic markers to identify synapses and correlate them with neuronal and neurite morphology.
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In this automated assay, translocation of the activated transcription factor from the cytoplasm to the nucleus is automatically detected and quantified for each cell. The assay is conveniently run using three detection channels, with a nuclear stain, a whole-cell stain, and a specific label for the transcription factor.
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Membrane integrity is a commonly measured parameter in cell viability assays. Cells with compromised membranes allow entry to otherwise cell-impermeant DNA-binding dyes, and this DNA staining serves as a cell death indicator that can be used as the basis for a single-channel assay. Invitrogen Image-iT DEAD Green Viability Stain (green; Cat. No. I10291) is an ideal dead cell stain because it will label the nuclei of dead cells and also survives fixation and permeabilization so that it can be readily multiplexed with other techniques.
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Image segmentation separates objects of interest (cells) from background or other features not relevant to the analysis and allows determination of items of interest in their appropriate spatial context such as translocation of biomarkers into the nucleus or changes in biomarker levels within particular cellular compartments. Key approaches for image segmentation involve using fluorescent stains selective for specific parts of the cell—nucleus, cytoplasm, plasma membrane, or other organelles—to delineate the whole cell.
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Reagents
Figure 2. The effects of cytochalasin D treatment on HeLa cell size as measured by HCS CellMask Blue stain. HeLa cells were treated with DMSO vehicle (left) or 10 µM cytochalasin D (right) for 3 hr before fixation and permeabilization. Samples were then labeled with HCS CellMask Blue stain, Alexa Fluor 488 dye–conjugated phalloidin to visualize filamentous actin (red), mouse anti–α-tubulin IgG (detected with Alexa Fluor 555 goat anti-mouse IgG, green), and TO-PRO-3 iodide to counterstain nuclei (magenta). The bar graph represents quantitative measurements of cell size as determined by HCS CellMask Blue stain to show the effects of cytochalasin D treatment.
Learn more about reagents for cell viability, proliferation, and function or reagents to determine cell structure.
For a more specific primary or secondary antibody, make your selection using these links:
High-throughput immunoassay screening enabled using the new CellInsight CX7 Pro Platforms
Comparison of the FirePlex™-HT no-wash immunoassay between the CellInsight CX7 and CX7 Pro instruments. The no-wash FirePlex assay optimized for high-throughput screening was performed according to the manufacturer’s protocol for detecting both 5 and 10 analytes per well. A 10-point standard curve was quantified to assay analyte reproducibility and dynamic range windows. The resulting assay was measured using either the CellInsight CX7 (left images) or CX7 Pro (right images) platforms and quantified using the FirePlex Analysis Workbench software. Only the CX7 Pro instrument (right images) was able to exceed 2.5 (15-bit) or 3.0 (16-bit) dynamic range and less than 15 percent intra-plate CVs. The significantly improved dynamic range and reproducibility performance is due to the CX7 Pro instrument's superior optics and 95% QE detection capability. This experiment was validated by Abcam™ using both 15- and 16-bit CX7 Pro sCMOS camera modes for HT-screening considerations.
Immuno-oncology application: Imaging antibody-dependent cell killing in breast cancer spheroids
Antibody-dependent cell killing in breast cancer spheroids. Human natural killer cells isolated with Dynabeads Untouched Human NK Cells Kit were labeled with CellTracker Deep Red dye. Human breast cancer cells (SKBR3) were grown overnight in Nunclon Sphera 96-well plates to form spheroids, treated with the anti-HER2 antibody Trastuzumab, then challenged with NK cells for 4 hours. Cells were stained with CellEvent Caspase-3/7 Green Detection Reagent and Hoechst 33342 and imaged on the CellInsight CX7 LZR High-Content Screening. Platform. The images are maximum intensity projection of multiple Z sections. CellEvent Caspase-3/7 Green Detection Reagent was used to study NK cell and antibody-mediated apoptosis in spheroids. CellTracker Deep Red was used to track NK cells within the spheroid. Trastuzumab bound to HER2 on SKBR3 cells amplifies the NK cell anti-tumor response via antibody-dependent cellular apoptosis.
Spheroid imaging and analysis application: segmentation and quantitation of EdU proliferation and spheroid size
Segmentation and quantitation of EdU and spheroid size using CellInsight CX7 LZR system. A549 cells were plated at a density of 5,000 cells per well on a Nunclon Sphera 96U-well microplate and incubated for 24 hours in a CO2 incubator. EdU was added at a final concentration of 10 µM and incubated for 1 hr. The spheroids were then washed and fixed with 4% formaldehyde and permeabilized with 0.25% Triton X-100. The spheroids were then stained for EdU using the Click-iT EdU Alexa Fluor 488 HCS Assay Kit following the kit protocol. The plate was imaged with a 4x objective using confocal mode on a CellInsight CX7 LZR High Content Screening Platform. The image is a maximum intensity projection of 200 optical z-slices of 1 micron each. Quantitation was performed with HCS Studio 2.0 software using the Morphology Explorer bio-application. The spheroid was segmented as one object, and EdU positive cells were counted as spots within the spheroid. Using Morphology Explorer bio-application, the spheroids were segmented as a whole object, and EdU positive cells were segmented inside the spheroid and number of cells was plotted.
Confocal imaging of 3D spheroids in live cell mode for bio-applications
Confocal imaging of 3D spheroids in live cell mode. (A) A549 cells were plated at a density of 5,000/well on a U-bottom plate and incubated for 48 hr in a CO2 incubator. Live spheroids were labelled for live and dead cells with a LIVE/DEAD Viability/Cytotoxicity Kit. The plate was automatically imaged with 10x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. Dead cells stained were observed in spheroid core (red) and live cells (green) were observed on the periphery of spheroids. (B) A549 cells were plated at a density of 5,000/well on a U-bottom plate and incubated for 24 hr in the CO2 incubator. Live spheroids were then stained with MitoTracker Orange CMTMRos and CellEvent Caspase-3/7 Green Detection Reagent for 30 min. The plate was automatically imaged with 10x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple Z sections. In A549 live spheroids, most of the cells are healthy as seen by MitoTracker Orange staining, and very few apoptotic cells were observed. (C) A549 cells were plated at a density of 5,000/well on a U-bottom plate and incubated for 24 hr in a CO2 incubator. Live spheroids were then stained with Image-iT Green Hypoxia Reagent for 30 min. The plate was automatically imaged with 10x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. Live A549 spheroids show hypoxia staining as stained with Image-iT Green Hypoxia Reagent. (D) SKBR3 cells were plated at a density of 5,000/well on a U-bottom plate and incubated for 24 hr in a CO2 incubator. Live spheroids were then incubated with pHrodo Red conjugated Herceptin antibody for 24 hr. The plate was automatically imaged with 4x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. The internalized pHrodo conjugated Herceptin antibody is observed in the intracellular vesicles. (E) Neurospheres were differentiated from neural stem cells (NSC) in Neurobasal Plus Medium with Culture One Supplement. Live neurospheres were then stained with Tubulin Tracker Deep Red for 1 hr. The plate was automatically imaged with 4X objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. Tubulin Tracker Deep Red stains the neurites in the neurospheres differentiated from NSC. (F) HeLa cells were plated at a density of 5,000/well on a U-bottom plate and incubated for 24 hr in a CO2 incubator. Activated T cells were labelled with CellTracker Deep Red and about 5,000 cells were added to each well. After 2 hr incubation with activated T cells, spheroids were stained with pHrodo Green AM Intracellular pH Indicator for 30 min. The cells were then washed 3x with PBS and imaged with 4x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. Intracellular increase in fluorescence of pHrodo Green AM upon addition of activated T cells.
Imaging and analyzing proliferating cells in spheroids
Analyzing proliferating cells in HeLa spheroids. HeLa cells were plated at a density of 5,000/well on a Nunclon Sphera U-bottom plate and incubated for 24 hr in a CO2 incubator. The spheroids were treated with 50 µM hydroxyurea for 24 hr. The spheroids were then pulsed with 10 µM 5-ethynyl-2’-deoxyuridine (EdU) for 30 mins. Spheroids were then washed with PBS and stained for proliferating cells using the Click-iT EdU Alexa Fluor 488 HCS Assay. Spheroids were then washed and stained with a Ki67 antibody conjugated to Alexa Fluor 647 dye. The spheroids were automatically imaged with 10x objective using confocal on a CellInsight CX7 LZR HCS instrument. The image is a maximum intensity projection of multiple z-sections. Spheroids were segmented as an object, and EdU- and Ki67-positive cells were quantitated as puncta within the spheroid. Both EdU- and Ki67-positive cells were seen in control spheroids. In hydroxyurea-treated spheroids, the actively proliferating s-phase cells disappeared (EdU negative) and only Ki67-positive cells were observed.
Immuno-oncology application: Imaging T cell penetration and killing of lung cancer spheroids
T cell penetration and killing of lung cancer spheroids. Spheroids were formed by seeding A549 cells using Gibco Minimal Essential Medium (MEM) in Nunclon Sphera U-bottom plates and culturing for 2 days. T cells isolated from human PBMCs using Dynabeads Human T-Expander CD3/CD28 were activated for 72 hr and labeled with CellTracker Deep Red Dye before adding to lung cancer spheroids for 4 hr. Cells were labeled with CellEvent Caspase 3/7 Reagent. T cell penetration and tumor cytotoxicity were evaluated using live-cell whole-spheroid imaging on the CellInsight CX7 LZR High-Content Platform. Activated T cells penetrated the spheroids (red) and induced apoptosis in target cells throughout the spheroids as seen by increased staining with CellEvent Caspase 3/7 Reagent (green).
Thermo Scientific high-content analysis instruments are cited extensively in reputable publications. HCA instrument citations in peer-reviewed publications are displayed here. HCA comprises a powerful combination of fluorescence microscopy, image processing, automated cellular measurements, and informatics tools that has enabled fundamental discoveries in basic research—and progression in drug compound discovery. See how researchers like you are using Thermo Scientific CellInsight High-Content Screening (HCS) Platforms to publish their results.
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