High Content Drug Discovery & Cancer Research

The development of new drugs and biopharmaceuticals will turn the science of today into the medicine of tomorrow. This requires a precise experimental implementation from researchers and manufactures as well. All steps to achieve that goal are associated with high time pressure and a lot of time consuming assay work – in a very broad range of applications.

Applications such as apoptosis monitoring, toxicity-studies, EC50 determination, surface marker analysis and ROS-detection represent only a part of the applications in which SYNENTEC actively supports your daily lab work with their fully automated high-throughput cell imagers CELLAVISTA® and NYONE®. Image cytometry - so fast that your cells don't even notice that they were out of the incubator.

SYNENTEC provides versatile ready-to-use solutions to discover the field of cancer research and drug development:

Apoptosis Monitoring, e.g.:

JC-1 Assay
AnnexinV-Assay
Cell Shrinking
Caspase Assay
TUNEL
BrdU

Toxicity Studies (EC50 / IC50) e.g.:

Wound Healing and Proliferation Assays
Live/Dead Assay
DNA-Damage with anti-γH2AX
Dose-Response Curves
NK-Cell mediated Lysis
Cell Migration and Proliferation

Cell Cycle/Mitosis e.g.:

pHH3-Assay
…

Immuno Cyto Chemistry & Multispectral Staining e.g.:

Antibody Multiplex Staining
Antibody Internalization
CD-Marker
Rare-Cell Analysis
Oxidative Stress (ROS) Detection
Characterizarion of 3D Tumor Spheroids

Caspase Assay – Extrinsic and Intrinsic Apoptotic Pathways

[Virtual Cytoplasm (1F)]

SYNENTEC’s imagers are able to analyze fluorescent caspase assays like the CellEvent™ Caspase-3/7 Detection Reagent. They can be combined with nuclear stains like Hoechst to quantify the percentage of caspase-positive cells. To detect necrotic or late apoptotic cells, a combination with markers entering only cells with compromised membranes (like propidium iodide or 7-AAD) is also possible.

Caspases are activated by the extrinsic and intrinsic apoptotic pathways. Their activity initiates many of the morphological and biochemical correlates of apoptosis, including DNA condensation and fragmentation, membrane blebbing and the formation of apoptotic bodies. Thus, assays detecting caspase activity are widely used to analyze apoptotic events in high-throughput screening.

SYNENTEC’s imagers are able to analyze fluorescent caspase assays like the CellEvent™ Caspase-3/7 Detection Reagent. They can be carried out in a variety of ways. For example, they can be combined with nuclear stains like Hoechst to quantify the percentage of caspase-positive cells. To detect necrotic or late apoptotic cells, a combination with markers entering only cells with compromised membranes (like Propidium Iodide or 7-AAD) is also possible.

Helpfull Image Analysis Applications of the YT-software (for further information look at AppNote High-content analysis of gemcitabine-induced caspase activity using NYONE® and YT®-Software)

Confluence non-invasively detects dose-dependent effects over time
Virtual Cytoplasm (1F) quantifies the cell nuclei and the percentage of caspase-positive cells
Image Statistics reliably analyzes e.g. a resazurin-based viability assay
…

Wound Healing Assay – Closure In Focus

[Wound Healing Assay]

Wound healing is a complex phenomenon conducted by numerous cells interacting with each other and their nearest neighbors when cell-cell contact is disrupted. This recovery is usually tracked with time lapse microscopy or can be monitored by imaging the sample at different time points.
In general, ready-to-us wound healing assays are easy to set up whereas manuell scratch assays are rather inexpensive but difficult to evaluate.

Wound healing assays are a convenient method to study various processes like cell polarization, cell migration and matrix remodeling. Furthermore, the role of certain factors or substances in wound healing, dose response analysis for cancer Treatment or even pathologic processes as cancer cell metastasis and invasiveness of tissues can be analyzed.
SYNENTEC® provides high-end automated microscop and image analysis tools. And CELLAVISTA® in combination with the Ibidi® incubation system provides also a way to perform wound healing and other live cell applications in real-time. Furthermore the high image quality allows documentation and evaluation of cell morphology.

Extract from the software readout for adherent cells (for further information look at AppNote Wound Healing Assay)

Wound dimension
Speed of wound closure
Time lapsed closure curve
…

Antibody Internalization - Below the Surface

[Confluence 1F / Suspension Cell Count 1F / Suspension Cell Count AB-Binding]

Internalized Antibody,
INTERNALIZATION ASSAY, ADC, INTERNALIZATION, ANTIBODY DISCOVERY, CELL CONFLUENCE, FLUORESCENCE IMAGING, CELL MORPHOLOGY, MDA-MB-468, CACO-2, CANCER RESEARCH

Antibody Internalization is a key parameter for high-content protein-based pharmaceuticals. Either in regards to antibody-drug conjugates (ADC) that have to be internalized to deliver cytotoxic small-molecule effectors to specific cells expressing a certain antigen. Or in regards to antibodies that have recruiting functions via their Fc-part to elicit ADCC or other cell based functions, where rapid internalization is not desirable. Also it is key to monitor internalization of antibodies that mediate receptor endocytosis to potentially downregulate receptor function and thus inhibit growth of tumor cells.

For information on internalization monitoring, please refer to the AppNote Antibody Internalization and see what's all possible with the high-NA lenses (here: 20x UPlanSAPO NA 0.75) and the highly sensitive sCMOS sensor from our scientific product line.

DNA-Damage Detection With Anti-γH2AX – X-Marks The Spot

[Nuclei Real Dot Count 1F]

The histone H2AX is a protein complexed in the nucleus of eukaryotes – the purpose of H2AX is to stabilize the genetic information (DNA). In addition H2AX has several functions in DNA repair and maintenance of chromosomes in the cell cycle. H2AX has also a medical significance as a laboratory value of DNA damage.

E.g. in response to DNA double strand-break H2AX is phosphorylated and then referred to gamma H2AX. The formation of γH2AX occurred even without exposure to exogenous noxae such as ionizing radiation. γH2AX is established as a sensitive detection of DNA double-strand breaks in science, especially in radiation biology. Here subnuclear structures are formed by accumulation, which appear as distinct points (gH2AX foci) after fluorescent staining and can be recognized with our automated imaging systems and be quantified with our integrated YT-software.

Extract from the software readout for suspension cells (for further information look at ShortNote Nuclei Real Dot Count 1F)

# of nuclei
# of γH2AX foci
% of foci positive nuclei
…

Mitochondrial Membrane Potential – Apoptosis Studies With JC-1

[JC-1 mito potential]

The programmed cell death (apoptosis) is a complex mechanism consisting of many particularly different meshing steps.
One early hallmark of apoptosis is the loss of mitochondrial membrane potential (delta psi_m; ΔΨm) resulting from a disruption of the mitochondrial membrane. To indicate the mitochondrial health in a suspension cell culture treated with H₂O₂, we used the cationic, lipophilic dye JC-1. This dye has the property to change the emitted fluorescence via accumulation and aggregation. In healthy cells JC-1 enters the negative charged mitochondrial matrix and enriches the mitochondria lumen where it builds J-aggregates after reaching a critical concentration. The J-aggregates are red fluorescent.
A collapse of ΔΨm, like in apoptotic cells, disables an accumulation of the JC-1 molecules. In those cells JC-1 remains in a monomeric, green fluorescent form. Therefore early apoptotic and healthy cells are easy to distinguish with fluorescence measurements by the NYONE® or CELLAVISTA® System.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 2F)

Total # of cells
% of Hoechst33342 and JC-green stained cells (=cells with loss of mitochondrial membrane potential
% of Hoechst33342 and JC-red stained cells (=viable cells)
…

AnnexinV – With Flip Flop On The Other Side

[Virtual Cytoplasm 2F]

These images show the difference between viable, apoptotic and dead cells
a) Shown are viable cells, the nuclei, stained with Hoechst. AnnexinV and PI could not bind. b) Shown are apoptotic cells with a green AnnexinV-FITC stained cell membrane and a blue Hoechst stained cell nucleus. c) Dead cells with PI (red) and Hoechst (blue) in the cell nucleus and a green membrane with AnnexinV-FITC. — These images show the difference between viable, apoptotic and dead cells a) Shown are viable cells, the nuclei, stained with Hoechst. AnnexinV and PI could not bind. b) Shown are apoptotic cells with a green AnnexinV-FITC stained cell membrane and a blue Hoechst stained cell nucleus. c) Dead cells with PI (red) and Hoechst (blue) in the cell nucleus and a green membrane with AnnexinV-FITC.

Another early marker of apoptosis is the translocation of phosphatidylserine (PS) in the lipid bilayer of apoptotic cells. In healthy cells PS is usually located on the inner side of the cell membrane. It is an active, enzyme dependent process to sustain this PS-asymmetry.
When the apoptosis cascade starts, the caspase-3 signal is given to stop the PS-asymmetry on the lipid bilayer, whereupon PS is translocated to the external membrane and serves as a recognition signal for phagocytes. On this early stage of apoptosis, AnnexinV conjugates have a direct access to the outer PS, even before loss of membrane integrity.
If AnnexinV is added to the cell culture it binds to all accessible PS-lipids. The FITC-labeled form of AnnexinV enables the fluorescent detection of apoptotic cells in the culture with SYNENTEC’s cell imager whereas the additional staining with Propidium Iodide helps to discriminate from dead/necrotic cells.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 2F)

# of only Hoechst33342-stained cells (=viable cells)
# of Hoechst33342 and AnxV stained cells (=apoptotic cells)
# of Propidium Iodide and AnxV stained cells (=necrotic cells)
…

Caspase Assays – Speed Up with Image Cytometry

[Virtual Cytoplasm 1F]

Apoptisis monitoring with multiple Caspase Assays

Caspases constitute a family of proteases responsible for the induction of controlled cell death. Upon activation, hydrolysis of the procaspase into active caspase occurs. Various caspases are among the critical enzymes in the apoptosis process and are well studied in the human organism. They are able to activate further procaseases and specifically hydrolyze cell substrates and structural proteins. The result of caspase activation is, among other things, the fragmentation of the cell, which ends in the formation of the 'apoptotic bodies'.

Due to the high degree of flexibility in channel selection, this assay can be carried out in a variety of ways with NYONE® and CELLAVISTA®. You can work with or without counter stain (Hoechst). You can use 2 differently fluorescent caspase detectors simultaneously. And the simultaneous use of propidium iodide or 7-AAD as necrosis detection is also possible.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 1F)

# of only Hoechst33342-stained cells (=viable cells)
# of Hoechst33342 and Caspase+ stained cells (=apoptotic cells)
% of Apoptotic Cells
…

Live/Dead Assay - The Binary System Of Life-Cycle

[Virtual Cytoplasm 2F]

The SYNENTEC Cytotoxicity Test is a three-color fluorescence assay designed to simultaneously measure the number of live, dead, and total cells. It is based on the enzymatic hydrolysis of cell permeant calcein AM by intracellular esterases in viable cells. Like esterase activity, membrane integrity is essential to the viability of cells. After cell death, membrane integrity is impaired. Since propidium iodide (PI) can not pass through the cell membrane of living cells, it is a good probe for dead cells.

Hoechst 33342 is used to compensate for variations in the number of cells seeded in each well. The received total number of cells allows normalization of the results per well for the control group. With this cytotoxicity assay, you can determine EC50 levels and create dose response curves in a high throughput manner.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 2F)

# of only Hoechst33342-stained cells (=total cells)
# of Hoechst33342 and Calcein stained cells (=viable cells)
# of Hoechst33342 and Propidium Iodide stained cells (=dead cells)
…

Cell Proliferation – Keep An Eye On It

[Confluence / Confluence 1F – Suspension Cell Count / Susp. Cell Count 1F]

Confluence monitoring is a useful tool to determine various properties of your cell line. SYNENTEC’s confluence image processing analysis is capable to solve a vast range of different questions – e.g. to monitor the growth rate under selective drugs or to prove toxicity by adding a drug library and monitor the growth rate over different concentrations as a first step of proving the influence of different drugs on the cell type of interest.

With SYNENTEC’s automated cell culture microscopes NYONE® and CELLAVISTA® and the included YT-image analysis software the proliferation analysis is possible for a variety of cells. Adherent lines can be detected as well as suspension cells. In addition, it is very easy to quantify additional fluorescent labels and their ratio to the total growth area.

The clearly arranged Time Chart option displays the data from various measurements of the same experiment. It displays all measured data in the appropriate plate format as line diagrams and thus gives a quick overview of the proliferation behavior per well.

Extract from the software readout for adherent cells (for further information look at ShortNote Confluence / Confluence 1F)

% confluence BF
% confluence FL
% ratio of confluence BF/FL
…

Extract from the software readout for suspension cells (for further information look at ShortNote Suspension Cell Count / Suspension Cell Count 1F)

# of cells in brightfield
# of cells in fluorescence
# of cells brightfield AND fluorescence
…

Cell Cycle And Mitosis – Lets Go Round And Round And Round

[pHH3 Assay]

Identification of mitotic status of cells can be important e.g. in cell production processes as well as for cancer related research on proliferation. With SYNENTEC’s imaging systems, mitotic cells can be automatically imaged and analyzed in a high throughput manner.

Before imaging, cells are stained with the nuclei dye Hoechst 33342 and immunohistochemically with a fluorescence labeled mitotic marker, e.g. α-pHH3 antibody. Samples are imaged in two fluorescence channels: one for Hoechst to determine the total number of cells and one for the subpopulation stained with the marker.

Mitotic cells and Hoechst stained cells can individually be quantified and evaluated very fast and precise with the integrated YT®-software in respect of fluorescence intensities, size and multiple other characteristics.

Extract from the software readout (for further information look at ShortNote pHH3 Assay)

Total Cell Count
pHH3 (=M-Phase) Cell Count
pHH3 (=M-Phase) M-Phase Cells [%]
Evaluated Area [mm²]
…

CD Marker – Immunological Staining Of Cluster Of Differentiation

[Suspension Cell Count 1F]

Cluster of differentiation (CD) are a large family of cell surface markers. The differentiation is conducted by functional and biochemical criteria. Their localization on the cell surface explains their main functions as cell adhesion proteins, signal transducer, receptors, activators, apoptosis initiators etc. and enables an easy immunological staining on living cells.

Since the CD markers are very cell type specific, there is a lot of ongoing research involved in the development of antibodies (AB) against CD-markers expressed by pathological cells. A good example is the monoclonal AB against B-cell marker CD20, which applies in B-cell derived lymphomas and leukemia but also in B-cell mediated autoimmune disorders.

A wide range of fluorophores for labeling the antibodies against the CD-marker of interest are supported by SYNENTEC’s imaging systems CELLAVISTA® and NYONE®. The samples will be measured with the 10x magnification, 1 brightfield channel, 1 fluorescent channel and will be analyzed with the “Suspension Cell Count 1F” operator of the YT-software®.

To check the fluorescence filter specifications of our systems visit the Fluorescence Viewer.

Extract from the software readout for suspension cells (for further information look at ShortNote Suspension Cell Count 1F)

# of cells in brightfield
# of cells in fluorescence
# of cells brightfield AND fluorescence
…

Rare Cell Analysis – Capturing Circulating Tumor Cells

[Rare Cells]

The analysis of the amount of circulating tumor cells (CTC) in the blood enables the controlling of a successful cancer therapy. The presence of those CTCs reflects a hint of the tumor growth and enables a method to monitor it. Unfortunately the concentration of CTCs in blood is quite low and to catch them is a rare event.

SYNENTEC’s imaging platforms (CELLAVISTA® and NYONE®) open a way to detect these rare events in a fast and reliable manner. The YT-Software® package uses embedded processing tools to pick the CTCs out of thousand blood cells to quantify them.

Extract from the software readout for suspension cells (for further information look at ShortNote Rare Cells)

Total # of detected rare cells
Average fluorescence intensity

Oxidative Stress – The Influence Of ROS

[Virtual Cytoplasm 1F]

Within the organism, reactive oxygen species (ROS) are constantly generated in the mitochondria as a product of cellular respiration (by monoamine oxidases and as part of the respiratory chain of complex I and complex III), but also by inflammatory cells in order to damage e.g. viruses and bacteria. ROS (especially hydrogen peroxide and nitric oxide) are also used in plant defense against pathogens.

The latest research results demonstrate that cancer cells exhibit increased intrinsic ROS stress due to increased metabolic activity, oncogenic stimulation and mitochondrial malfunction. Specifically abnormalities in mitochondrial DNA, as a major producer of ROS in cancer cells, are in the focus of the researchers.

Extract from the software readout for Virtual Cytoplasm 1F (for further information look at ShortNote Virtual Cytoplasm 1F)

Total # of cells
% of Hoechst33342 and ROS-stained cells
…

Quantification Of 3D Spheroids – Also Accumulations Are Separated

[Colony Count]

Spheroid Morphology Analysis & Quantification in Brightfield

Over the last years, it became obvious that the complex nature of cancer is not reflected in the widely used two-dimensional (2D) monolayer cell culture systems. Therefore, three-dimensional (3D) cell culture models became increasingly popular. One of these models is based on the formation of multicellular tumor spheroids.

Spheroids strikingly mirror the 3D cellular context of in vivo tumors. In order to become an alternative to 2D systems, it must be possible to generate 3D spheroids with a homogenous size to obtain comparable and reproducible results.

With the spheriod count image analysis operator of the YT-software it is possible to analys the spheroid morphology and to quantify each single spheroid, also if lying in accumulations, in brightfield channel.

Extract from the software readout for suspension cells (for further information look at ShortNote Colony Count)

Total # of Spheroids / well
Max. diameter per spheroid
Avg. spheroid diameter / well
...

High-Content Spheroid Analysis – Multiparametric Evaluation

[Spheroid Count]

High-Content 3D Spheroid in Kugelmeiers Spericalplate 5D

It has been sufficiently investigated that the complexity of tumor genesis is not reflected in conventional two-dimensional (2D) monolayer cell culture models. Therefore, three-dimensional (3D) cell culture models have gained interest in recent years. In these models, cells do not grow as one layer of cells but form a complex three-dimensional structure. This structure provides a more accurate representation of tumor physiology as occurring in cancer diseases.

Tumor-derived spheroids may prove to be instrumental for a high screening platform, for the investigation of cancer stem cells, or for the work with cancer stem cell-related tumor cells found in the circulation or body fluids.

One tool to produce such spheroids is the sphericalplate 5D invented by Kugelmeiers. This 24-well plate contains 750 round-bottomed microwells/ well with a high-end ultra-low attachment nanocoating which allows ideal cell aggregation and prevents the settlement of cells in other areas than the microcavities.

The SCIENTIFIC product line of CELLAVISTA® and NYONE® represents a very potent and easy-to-use way for investigating spheroid systems. It possible to perform high-content analyzes of 3D spheroids in high-throughput.

Extract from the software readout for Spheroids (for further information look at AppNote Kugelmeiers Sphericalplate 5D - Spheroid Imaging)

Total # of Spheroids / well
Max. diameter per spheroid
Avg. spheroid diameter / well
Brightfield and multichannel fluorescence collerations
Growth curves
Dose response validation
...