HCS image showing assay features for neurosphere

Explore various high-content screening (HCS) applications and high-resolution imagery that can be achieved using our automated and advanced HCS instruments/platforms. HCS or high-content analysis (HCA), reports quantitatively on parameters such as individual cell or organelle morphology and spatial distribution of targets. Explore high-content screening applications, performed by our scientists, ranging from 3D spheroid biology to 2D cell-based assays using our HCS platforms and reagents. Invitrogen fluorescent reagents are the preferred tools for labeling and detection of cellular targets. Thermo Scientific HCS Studio Cell Analysis Software is used with images from Thermo Scientific HCS platforms to build applications that answer complex biological questions.

Request a demoRequest a quoteRequest more info

Applications for Thermo Scientific HCS platforms

Advanced 3D Spheroids imaging and bio-applications with HCS platforms

Confocal imaging of 3D spheroids in live cell mode for bio-applications using CX7 LZR HCS instrument

Confocal imaging of 3D spheroids in live cell mode. (A) A549 cells were plated at a density of 5000/well on a U-bottom plate and incubated for 48 hrs in the 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then stained with MitoTracker Orange CMTMRos and CellEvent Caspase-3/7 Green Detection Reagent for 30 Mins. 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then stained with Image-iT Green Hypoxia Reagent for 30 Mins. 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then incubated with pHrodo Red conjugated Herceptin antibody for 24 hrs. 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 media 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Activated T cells were labelled with CellTracker Deep Red and about 5000 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 mins. 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.

Confocal imaging on CX7 LZR HCS system improves axial resolution of 3D spheroid imaging

Imaging antibody-dependent cell killing in breast cancer spheroids using CX7 LZR HCS instrument

Antibody-dependent cell killing in breast cancer spheroids. Human Natural Killer cells isolated with Dynabeads Untouched NK cells were labeled with CellTracker Deep Red. 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 four 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.

Imaging and analyzing proliferating cells in spheroids using CX7 LZR HCS instrument

Analyzing proliferating cells in HeLa spheroids. HeLa cells were plated at a density of 5000/well on a Nunclon Sphera U-bottom plate and incubated for 24 hrs in the CO2 incubator. The spheroids were treated with 50 µM Hydroxyurea for 24 hrs. 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. Spheroid were then washed and stained with a Ki67 antibody conjugated to Alexa Fluor 647 antibody. 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. Spheroid was 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, while in hydroxyurea treated spheroids, actively proliferating s-phase cells disappeared (EdU negative) and only Ki67 positive cells were observed.

Imaging and analysis of mitochondrial membrane potential using CX7 LZR HCS instrument

MitoTracker Orange CMXRos based quantitation of Mitochondrial membrane potential. Spheroids were formed by seeding A549 or HeLa cells using GIBCO minimal essential media (MEM) in Nunclon Sphera plates by culturing for 2days. DMSO vehicle or Niclosamide in incremental concentration was used to treat cells for 24 hours. Subsequently, cells were incubated for 30 minutes at 37°C with 2.5 µM CellEvent Caspase 3/7 Green Reagent and 250 nM MitoTra7cker Orange. Thermo Scientific CellInsight CX7 LZR High Content Imaging platform was used to capture confocal images and analyze the data using HCS Studio Software version 2.0. Niclosamide enhanced apoptosis (green) and lead to loss of mitochondrial membrane potential (orange) in a dose dependent fashion (top panel). Quantitative analysis of dose response Vs. mean signal intensity of orange and green can be seen in graphical format (bottom panel).

Measuring cellular health of spheroids with CyQUANT Direct, using CX7 LZR HCS instrument

Measuring cellular health of spheroids on CX7 LZR with CyQUANT Direct. A549 cells were plated in a Nunclon Sphera U-bottom plates with 5,000 cells per well.Cells were incubated at for 19 hours, to allow for spheroids to form. Spheroids were treated with different amounts of Gambogic Acid. At 48 hours, drug treated spheroids were stained with CyQUANT Direct and incubated at 37oC for 3 hours. Phase contrast images were obtained on the EVOS XL Core and the fluorescent images were obtained on the CellInsight CX7 LZR system. A549 spheroids are very compact with no drug treatment. Spheroids become lose and slightly larger in size with increasing concentrations of Gambogic acid. CyQUANT Direct signals are lower at higher concentrations of the drug due to toxicity.

T cell penetration and killing of lung cancer spheroids, imaging using CX7 LZR HCS instrument

T cell penetration and killing of lung cancer spheroids. Spheroids were formed by seeding A549 cells using GIBCO minimal essential media (MEM) in Nunclon Sphera U-bottom plates by culturing for 2 days. T cells isolated from human PBMCs using Dynabeads Human T-Expander CD3/CD28, were activated for 72 hours and labeled with CellTracker Deep Red before adding to lung cancer spheroids for four hours. 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 Analysis system. 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).

T cell mediated killing in HeLa spheroids, imaging using CX7 LZR HCS instrument

T cell mediated killing in HeLa spheroids. Spheroids were formed by seeding HeLa cells using Gibco minimal essential media (MEM) in Nunclon Sphera U-bottom plates by culturing for 2 days. T cells isolated from human PBMCs using Dynabeads Human T-Expander CD3/CD28, were activated for 72 hours and labeled with CellTracker Deep Red before adding to HeLa spheroids for four hours. 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 Analysis system. Activated T cells penetrated the spheroids (pink) and induced apoptosis in target cells throughout the spheroids as seen by increased staining with CellEvent Caspase 3/7 Reagent (green).

Segmentation and quantitation of EdU and spheroid size using CellInsight CX7 LZR system

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 the 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 done using HCS Studio 2.0 software using the Morphology Explorer bio-application. The spheroid was segmented as one object and then 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 using CX7 LZR HCS instrument

Confocal imaging of 3D spheroids in live cell mode. (A) A549 cells were plated at a density of 5000/well on a U-bottom plate and incubated for 48 hrs in the 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then stained with MitoTracker Orange CMTMRos and CellEvent Caspase-3/7 Green Detection Reagent for 30 Mins. 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then stained with Image-iT Green Hypoxia Reagent for 30 Mins. 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Live spheroids were then incubated with pHrodo Red conjugated Herceptin antibody for 24 hrs. 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 media 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 5000/well on a U-bottom plate and incubated for 24 hrs in the CO2 incubator. Activated T cells were labelled with CellTracker Deep Red and about 5000 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 mins. 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.

Confocal imaging on CX7 LZR HCS system improves axial resolution of 3D spheroid imaging

Imaging antibody-dependent cell killing in breast cancer spheroids using CX7 LZR HCS instrument

Antibody-dependent cell killing in breast cancer spheroids. Human Natural Killer cells isolated with Dynabeads Untouched NK cells were labeled with CellTracker Deep Red. 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 four 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.

Imaging and analyzing proliferating cells in spheroids using CX7 LZR HCS instrument

Analyzing proliferating cells in HeLa spheroids. HeLa cells were plated at a density of 5000/well on a Nunclon Sphera U-bottom plate and incubated for 24 hrs in the CO2 incubator. The spheroids were treated with 50 µM Hydroxyurea for 24 hrs. 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. Spheroid were then washed and stained with a Ki67 antibody conjugated to Alexa Fluor 647 antibody. 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. Spheroid was 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, while in hydroxyurea treated spheroids, actively proliferating s-phase cells disappeared (EdU negative) and only Ki67 positive cells were observed.

Imaging and analysis of mitochondrial membrane potential using CX7 LZR HCS instrument

MitoTracker Orange CMXRos based quantitation of Mitochondrial membrane potential. Spheroids were formed by seeding A549 or HeLa cells using GIBCO minimal essential media (MEM) in Nunclon Sphera plates by culturing for 2days. DMSO vehicle or Niclosamide in incremental concentration was used to treat cells for 24 hours. Subsequently, cells were incubated for 30 minutes at 37°C with 2.5 µM CellEvent Caspase 3/7 Green Reagent and 250 nM MitoTra7cker Orange. Thermo Scientific CellInsight CX7 LZR High Content Imaging platform was used to capture confocal images and analyze the data using HCS Studio Software version 2.0. Niclosamide enhanced apoptosis (green) and lead to loss of mitochondrial membrane potential (orange) in a dose dependent fashion (top panel). Quantitative analysis of dose response Vs. mean signal intensity of orange and green can be seen in graphical format (bottom panel).

Measuring cellular health of spheroids with CyQUANT Direct, using CX7 LZR HCS instrument

Measuring cellular health of spheroids on CX7 LZR with CyQUANT Direct. A549 cells were plated in a Nunclon Sphera U-bottom plates with 5,000 cells per well.Cells were incubated at for 19 hours, to allow for spheroids to form. Spheroids were treated with different amounts of Gambogic Acid. At 48 hours, drug treated spheroids were stained with CyQUANT Direct and incubated at 37oC for 3 hours. Phase contrast images were obtained on the EVOS XL Core and the fluorescent images were obtained on the CellInsight CX7 LZR system. A549 spheroids are very compact with no drug treatment. Spheroids become lose and slightly larger in size with increasing concentrations of Gambogic acid. CyQUANT Direct signals are lower at higher concentrations of the drug due to toxicity.

T cell penetration and killing of lung cancer spheroids, imaging using CX7 LZR HCS instrument

T cell penetration and killing of lung cancer spheroids. Spheroids were formed by seeding A549 cells using GIBCO minimal essential media (MEM) in Nunclon Sphera U-bottom plates by culturing for 2 days. T cells isolated from human PBMCs using Dynabeads Human T-Expander CD3/CD28, were activated for 72 hours and labeled with CellTracker Deep Red before adding to lung cancer spheroids for four hours. 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 Analysis system. 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).

T cell mediated killing in HeLa spheroids, imaging using CX7 LZR HCS instrument

T cell mediated killing in HeLa spheroids. Spheroids were formed by seeding HeLa cells using Gibco minimal essential media (MEM) in Nunclon Sphera U-bottom plates by culturing for 2 days. T cells isolated from human PBMCs using Dynabeads Human T-Expander CD3/CD28, were activated for 72 hours and labeled with CellTracker Deep Red before adding to HeLa spheroids for four hours. 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 Analysis system. Activated T cells penetrated the spheroids (pink) and induced apoptosis in target cells throughout the spheroids as seen by increased staining with CellEvent Caspase 3/7 Reagent (green).

Segmentation and quantitation of EdU and spheroid size using CellInsight CX7 LZR system

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 the 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 done using HCS Studio 2.0 software using the Morphology Explorer bio-application. The spheroid was segmented as one object and then 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.