HCS and HCA Sample Data

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).