The proof of the performance is in the data

Clear, detailed publication-quality images and quantitative data are what the CellInsight High-Content Screening platforms are engineered for. Review sample data, including fluorescence images, graphs, and videos for life sciences applications such as angiogenesis, apoptosis, autophagy, cell cycle and proliferation, endocytosis, and viability.


Images obtained from the CellInsight CX7 LZR platform

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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.

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Segmentation and quantitation of EdU 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.

live-cell-confocal-imaging-3d-spheroids-using-hcs-system-1000x738

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 neurtites 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.

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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.

imaging-lung-cancer-spheroids-killing-penetration-T-cells-1000x392

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).

Images obtained from the CellInsight CX7 platform

HeLa cells imaged using the CellInsight CX7 platform

Multiparameter HeLa cell imaging

HeLa cell image acquired using the  CX7 HCS platform and stained with HCS NuclearMask Blue (blue), MitoTracker Orange (green), and Alexa Fluor 647 Phalloidin (red).

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Multiparameter A549 cell imaging

A549 cell image acquired using the CX7 HCS platform and stained with HCS CellMask Blue (blue), Alexa Fluor 488 Phalloidin (green), and Alexa Fluor 750 secondary (pink).

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H&E brightfield imaging

Three-color brightfield image of fixed human breast cancer tissue stained with KI67-DAB, counterstained with hematoxylin and eosin, and imaged.

Images obtained from the CellInsight CX5 platform

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Multiparameter BPAE cell imaging

BPAE cells imaged using the CX5 HCS platform and stained with DAPI (blue), Alexa Fluor 568 Phalloidin (red), and Alexa Fluor 488 secondary (green).

HeLa cells imaged with CellInsight CX5

Multiparameter HeLa cell imaging

HeLa cells imaged using the CX5 HCS platform and stained with Hoechst 33342 (blue) and Alexa Fluor 680 phalloidin (yellow).



Sample applications

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.

 

Automatically measured properties: 

  • % connected tubes
  • Mean connected tube area
  • Mean connected width
  • Mean connected tube node spacing
  • Angiogenic index

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield or confocal
  • 5x magnification

Protocols and reagents:

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.

Suramin serves as a potent inhibitor of angiogenesis.

A dose-response for suramin concentration can be plotted against a variety of properties measured automatically using Thermo Scientific HCS platform with Thermo Scientific HCS Studio software and plotted using GraphPad Prism software.

Graph of five different angiogenesis assay parameters

Caspase assay for HCS that allows the simple detection of active caspases in living cells in real time or in fixed cells.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield or confocal
  • 10x or 20x magnification

Protocols and reagents:

Hap1 cells were labeled with CellEvent Caspase-3/7 Green reagent and Hoechst 33342 following treatment with staurosporine. The cells were imaged using a Thermo Scientific 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 CellEvent green fluorescence from each cell.

HCS Apoptosis Caspase Assay

Dose-dependent increase in the induction of apoptosis, as reported by activation of Caspase 3/7. The sensitivity of two cell types: wild type Hap1 (red) and Hap1 lacking ATG5 (blue), were compared.

Line graph of mean signal intensity vs staurosporine concentration

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.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield or confocal
  • 20x magnification

Protocols and reagents:

HeLa cells treated to induce apoptosis and imaged. Untreated (Left) and staurosporine-treated (Right) HeLa cells stained using the Invitrogen Molecular Probes Click-iT TUNEL Alexa Fluor 594 Imaging Assay, for Microscopy & HCS and Invitrogen Molecular Probes Hoechst 33342. Cells were imaged using a Thermo Scientific HCS instrument, and fluorescence intensities and count ratios were measured using Thermo Scientific HCS Studio Software.

Two-panel HCS image of nonapoptotic and apoptotic cells

Dose-response plots for a variety of compounds and cell types using Invitrogen Molecular Probes Click-iT TUNEL Alexa Fluor 647 to measure apoptosis. Responses were measured automatically using Thermo Scientific HCS platform with Thermo Scientific HCS Studio 2.0 Cell Analysis Software and plotted using GraphPad Prism software.

Four-panel graph of data for different angiogenesis assays

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.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 20x magnification

Protocols and reagents:

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.

Dose-response plot of LC3B granules per cell with increasing chloroquine concentration.

Graph of granules vs chloroquine concentration

Dose-response plot of LC3B fluorescence intensity with increasing chloroquine concentration.

Graph of fluorescence vs chloroquine concentration

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.

 

Automatically measured properties: 

  • DNA content
  • Intensity levels of up to 3 secondary targets
  • Correlation at single-cell and population level

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 10x magnification

Protocols and reagents:

Cell cycle assay. A549 cells were treated with increasing doses of a mitotic inhibitor for 24 hours. The cells were stained for phospho Histone H3 and detected using an Invitrogen Alexa Fluor 488 secondary antibody. Invitrogen HCS NuclearMask Deep Red stain was used as a nuclear segmentation tool.

Two-panel HCS image showing cell cycle assay results on control vs nocodazole treated cells

Dose-response plot of mitotic inhibitors in A549 cells using a non-linear regression with GraphPad Prism software to determine an EC.

Two-panel graph showing effect of different mitotic inhibitors

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.

 

Automatically measured properties: 

  • DNA content
  • Intensity levels of up to 3 secondary targets
  • Correlation at single-cell and population level

Imaging mode: 

  • Total cells
  • Replicating cells

Protocols and reagents:

A549 cell proliferation assay. A549 cells were treated with increasing doses of paclitaxel. Cells were stained with Invitrogen Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (red) and Invitrogen Hoechst 33342 stain (blue). Cells were imaged using the Thermo Scientific CellInsight CX7 High-Content Analysis Platform, and total cells and replicating cells were measured using Thermo Scientific HCS Studio Software.

 

Multipanel HCS images showing the effect of paclitaxel on proliferation

Dose-response plots of U2OS cells treated with increasing concentrations of proliferation inhibitors and stained using Invitrogen Molecular Probes Click-iT Plus EdU labeled with Invitrogen Molecular Probes Alexa Fluor 647 dye.

Two-panel graph showing proliferation vs increasing inhibitor concentration

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.

 

Automatically measured properties: 

  • Whole cell shape and dimensions
  • Number of cytoskeletal fibers, their dimensions, and alignment
  • Intracellular arrangement, location, and texture measurements of cytoskeletal labels

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms- second image was imaged using the CX7 platform
  • Widefield, 20x or 40x magnification

Protocols and reagents:

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.

Cellular dose-response to cytochalasin as shown by cell area, nuclear intensity, actin fiber count, tubulin fiber count, and actin fiber intensity.

Graph of five different cytoskeletal rearrangement assay parameters

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.

 

Automatically measured properties: 

  • Whole cell shape and dimensions
  • Number of cytoskeletal fibers, their dimensions, and alignment
  • Intracellular arrangement, location, and texture measurements of cytoskeletal labels

Imaging mode: 

  • DNA damage
  • Cytotoxicity

Protocols and reagents:

Using the HCS DNA Damage Kit, researchers can detect and quantitate DNA damage and changes in cell permeability.

Graphical representation of the DNA damage assay experiment

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.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms- second image was imaged using the CX5 platform
  • Widefield or confocal
  • 20x magnification

Protocols and reagents:

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).

Dose-dependent loss of EGF receptor (green trace) and EGF internalization (red trace) with increasing concentrations of Pitstop 2 inhibitor (Abcam, Inc.) in U2OS cells. At high concentrations of Pitstop 2 inhibitor, cells are unable to internalize the EGF receptor and its ligand.

HCS Endocytosis Assay sample data

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.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values of spots in different cell regions

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms

Protocols and reagents:

  • See selection guides below

Lipid vesicles in bone marrow—derived hMSCs visualized with LipidTOX Green neutral lipid stain and counterstained with Hoechst 33342

Microscopic view of HCS Lipidtox assay results  showing staining of neutral lipid stains and phospholipids

Selection guides for HCS LipidTOX stains

Steatosis/Adipogenesis

  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
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

Steatosis/Phospholipidosis

  HCS LipidTOX Phospholipidosis and Steatosis Detection Kit HCS LipidTOX Phospholipidosis and Steatosis Detection Kit
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

Phospholipidosis/Adipogenesis

  HCS LipidTOX Green Phospholipidosis Detection Reagent HCS LipidTOX Red Phospholipidosis Detection Reagent
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.

 

Automatically measured properties: 

  • Mitochondrial membrane potential
  • Cell viability
  • Nuclear shape and dimensions

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 10x or 20x magnification

Protocols and reagents:

HepG2 following Hoechst 33342 staining to provide nuclear segmentation for automated cell counting and nuclear morphology measurement.

HCS image showing fluorescent  staining of nuclei and other cell structures

Image-iT DEAD Green stain used to label dead cells and provide a viability determination based on cell membrane permeability.

HCS image showing fluorescent staining of dead cells

MitoTracker Orange labels mitochondria with polarized membranes, with signal intensity proportional to membrane potential and mitochondrial health.

HCS image of fluorescenct cells showing nuclear segmentation and mitochondrial membrane potential

Dose-response plot for HepG2 cells treated with increasing concentrations of valinomycin. Cells were stained with Invitrogen MitoTracker Orange to measure mitochondrial membrane potential, Invitrogen Hoechst 33342 dye to count total cells based on nuclear intensity, and Invitrogen Image-iT DEAD Green to identify dead cells. Automated imaging and analysis was done using Thermo Scientific HCS Platform and the data were used to calculate EC50 values for cell loss, mitochondrial membrane potential, and membrane permeability using GraphPad Prism software.

Graph of three different mitochondrial assay parameters

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.

 

Automatically measured properties: 

  • Cell count
  • Cell size
  • Neurite count
  • Neurite length
  • Neurite branching
  • Since analysis is performed during image acquisition, it is possible to acquire an optimized data set to ensure the number of selected neurons to satisfy your statistical needs.

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield or confocal
  • 10x magnification

Protocols and reagents:

Primary neuron outgrowth assay.(Right) A hippocampal primary neuron preparation was labeled using Invitrogen Hoechst 33342 (blue) and an Alexa Fluor 647 immunoconjugate. (Right) Neurite outgrowth features were automatically identified using Thermo Scientific HCS Studio Software. Cell bodies are identified in blue, and neurons are identified in green.

Two-panel HCS neurite outgrowth assay images showing stained cells and software overlay

Dose-response plots showing the effect of glutamate and kainite concentration on rat hippocampal neurons using automated measurements of neuron number, neurite count, neurite length, and neurite intensity.

Four-panel graph of different neurite outgrowth assay parameters

Using fluorogenic reporters such as CellROX dyes allows for the real-time analysis of ROS generation in living cells and in fixed-cell preparations.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms- second image was imaged using the CX5 platform
  • Widefield or confocal
  • 10x or 20x magnification

Protocols and reagents:

Hap1 cells were treated with menadione and labeled with CellROX Green and Hoechst 33342The cells were imaged using a Thermo Scientific 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 CellROX green fluorescence from each cell.

Dose-dependent increase in ROS production in Hap 1 cells loaded with CellROX Green and treated with menadione for one hour.

HCS Oxidative Stress Assay sample data

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.

 

Automatically measured properties: 

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms- second image was imaged using the CX5 platform
  • Widefield or confocal
  • 10x or 20x magnification

Protocols and reagents:

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.

Dose-dependent decrease in protein synthesis following treatment of cells with cyclohexamide. The sensitivity of two cell types: wild type Hap1 (red) and Hap1 lacking ATG5 (blue), were compared.

HCS Protein Synthesis sample data

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).

 

Automatically measured properties: 

  • Nuclear mask identification
  • Spots identified and localized by channel

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 10x magnification

Protocols and reagents:

Immunofluorescent detection of Oct4 (green) and NKx2.5 (red) to determine differentiation status in H9 cells stained with DAPI (blue).

Three-panel HCS image showing stem cell marker appearance over the assay course

Identifying differentiated stem cells. DAPI staining is used to automatically generate a nuclear mask (blue outline), then cells positive for the differentiation marker (in this case Oct4) within the nuclear region are highlighted in red.

Two-color HCS image showing nuclei and Oct4 location

Results from stem cell differentiation assay. The percentages of Oct4+ and Nkx2.5+ nuclei were quantified in cells fixed at the indicated time points. Prior to differentiation (Day 0), nearly 100% of all cells were Oct4+/Nkx2.5–, consistent with a pluripotent state. At Day 6, greater than 90% of cells analyzed were co-positive for both markers. After day 6, the frequency of Oct4+ cells declined, consistent with a loss of pluripotency and a transition to a terminally differentiated cardiomyocyte phenotype.

Bar chart showing two stem cell marker measurements over time

This automated assay uses the co-localization of presynaptic and postsynaptic markers to identify synapses and correlate them with neuronal and neurite morphology.

 

Automatically measured properties: 

  • Neurite count
  • Neurite length
  • Neurite branching
  • Presynaptic vesicles
  • Postsynaptic spots
  • Synapse number
  • Since analysis is performed during image acquisition, it is possible to acquire an optimized data set to ensure the number of selected neurons to satisfy your statistical needs.

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield or confocal
  • 20x magnification

Protocols and reagents:

Syantogenesis assay.(Left) A hippocampal primary neuron preparation was labeled using Invitrogen Hoechst 33342 stain (blue) and an Invitrogen Alexa Fluor 488 MAP2 antibody conjugate. (Center and Right) Synaptogenesis features were automatically identified using Thermo Scientific HCS Studio Software. Cell bodies are identified in blue, and spots are identified in pink. Neurites are identified in green, and overlaps between spots and neurites are labeled in yellow.

Three-panel HCS synaptogenesis assay image showing stained cells and software overlays

Dose-response plots showing the effect of glutamate and kainite concentration on rat hippocampal neurons using automated measurements of presynaptic intensity, postsynaptic intensity, vesicle counts, and synapse counts.

Four-panel graph of different synaptogenesis assay parameters

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.

 

Automatically measured properties: 

  • Transcription factor intensity in the cytoplasm and in the nucleus
  • Difference in transcription factor intensity between the cytoplasm and nucleus, as a measure of cytoplasm-to-nucleus translocation
  • Ratio of transcription factor intensity between the cytoplasm and nucleus, as a measure of cytoplasm-to-nucleus translocation
  • Percentage of cells in the well above a user-definable threshold for the nucleus-to-cytoplasm intensity difference or ratio for one or more targets
  • Nuclear shape and dimensions

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 10x or 20x magnification

Protocols and reagents:

Transcription factor activation(Left) HeLa cells were treated with TNFα, fixed, permeabilized, and stained with Invitrogen HCS NuclearMask Blue (blue), and the NFκB was labeled by indirect immunofluorescence (green). (Right) Cell features for the assay were automatically detected using Thermo Scientific HCS Studio Software: nucleus (blue overlay), cytoplasm (green overlay), and NFκB intensity. The red overlay shows the cells where translocation did not occur.

Two-panel HCS NFκB translocation assay image showing stained cells and software overlay

NFκB cytoplasm-nucleus intensity difference vs. ratio. NFκB intensity differences between the cytoplasm and nucleus were automatically calculated and plotted. Red dots in the scatter plot of the NFκB cytoplasm-nucleus intensity difference versus ratio correspond to cells that did not exhibit NFκB translocation.

Graph of measured NFκB translocation parameters

Dose-response plot showing the activation of NFkB by increasing TNFα concentration. The y-axis shows the intensity difference between the nucleus (containing translocated NFkB) and the surrounding cytoplasm (containing un-translocated NFkB) as a measure of NFkB translocation.

Graph showing relationship between NFκB activation and TNFα concentration

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.

 

Automatically measured properties: 

  • Single-channel
  • Two-channel
  • Total cells
  • Live cells
  • Dead cells

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms
  • Widefield
  • 10x magnification

Protocols and reagents:

Single-channel viability assay with multiplexing. HeLa cells treated with camptothecin and stained with Invitrogen Hoechst 33342 (blue channel) for a total cell count. Dead cells are stained with Invitrogen Image-iT DEAD Green (green channel) and proliferating cells are labeled using Invitrogen Click-iT PLUS EdU with Invitrogen Alexa Fluor 647 picolyl azide (deep red channel).

Eight-panel HCS image showing 3 viability parameters at 2 camptothecin concentrations

Two-channel viability assay. A549 cells were treated with varying concentrations of camptothecin and labeled with the Invitrogen LIVE/DEAD Cell Imaging Kit. Living cells sequester the LIVE Green probe, whereas dead cells become permeable to the DEAD Red stain. Cells were counterstained with Invitrogen Hoechst 33342 for total cell count.

Microscopic view of A549 cells stained in 3-color fluorescence, indicating live vs dead cells

Single-channel viability assay with multiplexing. Dose-response curve for HeLa cells treated with camptothecin, stained with Invitrogen Image-iT DEAD Green and measured using the Thermo Scientific CellInsight CX5 High Content Screening Platform.

Graph of dead cells vs camptothecin

Two-channel viability assay. Dose-response plot showing the effect of camptothecin concentration on cell viability using the Invitrogen LIVE/DEAD Cell Imaging Kit.

Graph of two viability parameters

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.

 

Automatically measured properties: 

  • Nuclear
  • Cytoplasmic
  • Plasma membrane
  • Organelle

Imaging mode: 

  • CellInsight CX5 and CX7 Platforms 
  • Widefield
  • 10x magnification

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 stainAlexa 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.

Figure 3. Plasma membrane image segmentation of live cells using CellMask Deep Red plasma membrane stain. HaCaT cells (an immortalized human keratinocyte cell line) transfected with a mitochondrion-targeted eGFP (pseudocolored green) construct were incubated with 1 µM Hoechst 33342 (pseudocolored blue) and then 8 μg⁄mL CellMask Deep Red plasma membrane stain (pseudocolored red). Cells were imaged with a Zeiss Cell Observer HS microscope. The image stack was deconvolved with the Zeiss AxioVision 3D Deconvolution module with the point spread function acquired using a 200 nm TetraSpeck fluorescent microsphere. Image submitted by Christian Junker, University of Saarland, Department of Biophysics, Homburg, Germany.


Secondary antibodies and recommended assays by instrument

Thermo Fisher Scientific is continuously expanding their selection of fluorochromes, fluorochrome-conjugated secondary antibodies, and assays. Visit the HCS Reagent Selection Guides for the latest compatible instruments by model. 


Reference articles

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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