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Drugs & Cancer

Research of tumor formation, metastases, tumor reduction and cancer prevention are major achievements of our society today.

Introduction

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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®.

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

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

  • Live/Dead Assay
  • DNA-Damage with anti-γH2AX
  • NK-Cell mediated lysis
  • Cell Proliferation
  • Wound Healing

Cell Cycle/Mitosis e.g.:

  • pHH3-Assay

Immuno Cyto Chemistry e.g.:

  • Antibody Multiplex Staining
  • CD-Marker
  • Rare-Cell Analysis
  • Oxidative Stress (ROS) detection

Mitochondrial Membrane Potential – Apoptosis Studies With JC-1

[JC-1 mito potential]

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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 psim; 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 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (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.

Schematic illustration of JC-1 monomers (green dots) and J-aggregates (red dots) depending on the mitochondrial membrane potential.<br/>In healthy cells mitochondria are in an energized condition with a polarized mitochondrial membrane (a). Part of the apoptotic pathway is the uncoupling of the proton gradient of mitochondria and a loss of membrane integrity. This has the consequence that the cationic JC-1 dye gets no longer actively enriched in the mitochondrial lumen and therefore it cannot aggregate (b).
Schematic illustration of JC-1 monomers (green dots) and J-aggregates (red dots) depending on the mitochondrial membrane potential.
In healthy cells mitochondria are in an energized condition with a polarized mitochondrial membrane (a). Part of the apoptotic pathway is the uncoupling of the proton gradient of mitochondria and a loss of membrane integrity. This has the consequence that the cationic JC-1 dye gets no longer actively enriched in the mitochondrial lumen and therefore it cannot aggregate (b).

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<br/>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.

Simplified scheme of the difference between viable, apoptotic and dead cells<br/>a) Shown is a viable cell, phosphatidylserine is only in the inner leaflet of the bilayer, AnnexinV-FITC cannot bind, PI stays outside the cell. b) Shown is a apoptotic cell, PS has moved to the outside of the membrane, AnnexinV binds, PI stays outside. c) Shown is a dead cell, the membrane is permeable, PI can get into the cell and stains the cell nucleus, AnnexinV-FITC binds to the inner and outer leaflet.
Simplified scheme of the difference between viable, apoptotic and dead cells
a) Shown is a viable cell, phosphatidylserine is only in the inner leaflet of the bilayer, AnnexinV-FITC cannot bind, PI stays outside the cell. b) Shown is a apoptotic cell, PS has moved to the outside of the membrane, AnnexinV binds, PI stays outside. c) Shown is a dead cell, the membrane is permeable, PI can get into the cell and stains the cell nucleus, AnnexinV-FITC binds to the inner and outer leaflet.

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)
  •  …

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

[Virtual Cytoplasm 2F]

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The SYNENTEC Cytotoxicity Assay is a three color fluorescence assay to simultaneously determine the numbers of live, dead and total cells. It is based on the enzymatic hydrolyzation of the cell permeant Calcein AM (Calcein acetoxymethyl ester) by intracellular esterases in viable cells. 
Just like esterase activity, membrane integrity is essential for the viability of cells. After cell death the membrane integrity is affected. As Propidium Iodide (PI) cannot pass the cell membrane of living cells, it is a good probe for dead cells. 

Hoechst 33342 (2,5'-Bi-1H-Benzimidazol) is used to compensate for variations in the number of cells seeded in the individual wells. The received total number of cells allows the normalization of the results per well to the control group.
With this cytotoxicity assay you are able to determine EC50 values e.g. for DMSO (dimethyl sulfoxide) in mammalian cell line with the NyONE® or the Cellavista® system in a high throughput manner.

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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)

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

[Nuclei Real Dot Count 1Fs]

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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 Suspension Cell Count 1F)

  • # of nuclei
  • # of γH2AX foci
  • % of foci positive nuclei

Cell Proliferation – Keep An Eye On It

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

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 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.

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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

Wound Healing Assay – Closure In Focus

[Wound Healing Assay]

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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 points in time (as shown above). 
In general, wound healing assays are easy to set up and are rather inexpensive. Thus they 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 or even pathologic processes as cancer cell metastasis and invasiveness of tissues can be analyzed. 
The Cellavista® provides a high-end automated microscopy and image analysis tools and in combination with the Ibidi® incubation system also a way of performing 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 ShortNote Wound Healing Assay)

  • Wound dimension
  • Speed of wound closure
  • Time lapsed closure curve

  •  

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

[pHH3 Assay]

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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.

Cell cycle and mitosis with pHH3 gradient<br/>Cell cycle (left) showing mitosis and cytokinesis as well as interphase (G1-S-G2) and G0 resting phase. Mitosis (right) consists of pro-, prometa-, meta-, ana- and telophase. The mitosis marker pHH3 can be detected starting from late G2-phase with its maximum intensity at metaphase of mitosis. PHH3 signal starts to disappear with the beginning of anaphase.
Cell cycle and mitosis with pHH3 gradient
Cell cycle (left) showing mitosis and cytokinesis as well as interphase (G1-S-G2) and G0 resting phase. Mitosis (right) consists of pro-, prometa-, meta-, ana- and telophase. The mitosis marker pHH3 can be detected starting from late G2-phase with its maximum intensity at metaphase of mitosis. PHH3 signal starts to disappear with the beginning of anaphase.

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]

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 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 Retuximap, a 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]

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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 ingtensity

Oxidative Stress – The Influence Of ROS

[Confluence 1F / Suspension Cell Count 1F]

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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