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Mechanism of Action Studies, Assay Multiplexing and other Procedures  

Toxicity accounts for about 40-45% drug attrition rates with lack of efficacy accounting for about 20-25% attrition. When cytotoxicity occurs, it is often necessary to know the mechanism of action that causes cytotoxicity.

 

HemoGenix® divides cell-based assays into those that affect the proliferation or differentiation processes, since a single assay cannot measure both processes using the same readout. To study both proliferation and differentiation processes, assays often have to be multiplexed with each other so that information pertaining to both processes can be obtained from the same sample. Multiplexing also allows the mechanism of action to be determined. Virtually every assay that has been developed by HemoGenix® has been designed with multiplexing in mind. In this way, it is possible to provide our clients with the most amount of information possible from a single sample so that the most highly informed decisions can be made.

 

The list below of mechanism of action and multiplexing assays provides the reader with the most important type of assays that can be used in combination with HALO®, ImmuneAssays™, MSCGlo™, STEMAssays™, XVPrime-Assays™ and CLAssays™. For more information or whether HemoGenix® can perform an assay that is not in the list, please contact us directly.

 

Further down this page are other procedures and assays that are basic to cell-based studies. In addition, target cells might need to be expanded and/or purified. This is especially the case for stem cell populations. A list (not exclussive) of these procedures is also provided below. 

 

Mechanism of Action and Multiplexing Assays

  • Cell count
  • Dye exclusion viability
  • Metabolic viability (LIVEGlo™)
  • Cell and colony imaging
  • Image analysis
  • MTT absorbance assay
  • MTS absorbance assay
  • XTT absorbance assay
  • BudR assay
  • CellQuant® fluorescence assay
  • Growth factor/cytokine production assays
  • Growth factor/cytokine release assays
  • Real time chemotaxis / cell migration
  • Fluorescence assays
  • Intracellular antigen expression assays by flow cytometry
  • Oxidative DNA damage by flow cytometry using OxyFLOW™ (see below)
  • Extracellular membrane antigen expression by flow cytometry
  • Cell cycle analysis
  • Megakaryocyte ploidy analysis
  • Apoptosis by Annexin/propidium iodide and flow cytometry
  • Apoptosis by TUNEL
  • Apoptosis by caspase (2, 6, 3/7, 8, 9) detection (luminescent)
  • Apoptosis by ADP:ATP ratio
  • Mitochondrial dysfunction assays
  • Phosphodiesterase assays
  • Kinase assays
  • Protease assay (luminescent)
  • Lactate dehydrogenase (LDH) assays
  • Glutathione assay (luminescent, oxidative stress)
  • P-Glycoprotein assay (luminescent)
  • Monoamine oxidate (MOA) assay (luminescent)
  • UDPglucuronosyltransferase (UGT) assay (luminescent)
  • Cytochrome P450 luminescence assays: 1A2, 2C9, 3A4, 2C19, 2D6 for drug interaction 

 

Oxidative DNA Damage (OxyFLOW™)

A number of disease or pathological states can be attributed to or involve oxidative DNA damage. These include, but are not limited to:

  • Arthritis
  • Stroke
  • Ischemia-reperfusion therapy
  • Neurodegenerative disorders
  • Inflammation
  • Crohn's disease
  • Multiple sclerosis
  • Teratogenesis
  • Carcinogenesis


In addition, a number of biological processes have mechanisms related to or directly involved with oxidative DNA damage. These include:

  • Aging
  • Normal metabolic activity
  • Drug therapy
  • Oxidative stress
  • Nutrient deficiency
  • Genotoxicity
  • Radiation
  • Xenobiotics


Oxygen free radicals are highly active species that cause significant damage to DNA. One of the products is 8-oxoguanine. OxyFLOW™ is a flow cytometric assay for cell suspensions derived from non-adherent or adherent cells that detect 8-oxoguanine adducts as a result of oxidative DNA damage. A fluorescein isothiocyanate (FITC) - conjugated 8-oxoguanine binding protein is used to detect the presence of these adducts. 

 

 


Measurement of oxidative DNA damage using OxyFLOW and flow cytometry

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OxyFLOW™ - Assay Principle

The target cells are prepared into a single cell suspension. The cells are first subjected to permeation followed by incubation with the FITC-conjugated binding protein. The binding protein enters the cells and binds to 8-oxoguanine adducts. After washing the cells, they are suspended in buffer and analyzed by flow cytometry. Target cells are also used to perform the methylene blue standard curve which is performed in parallel. By incubating cells with methylene blue and exposing them to bright light, single-standed DNA damage occurs. With increasing doses of methylene blue, there is an increase in fluorescence intensity, indicating a concomitant increase in oxidative DNA damage. At high doses, the fluorescence intensity decrease. This is an indication of cell death due to oxidative DNA damage. An example of a methylene blue standard curve is shown in the diagram to the left.

 

 


Correlation between oxidative DNA damage using OxyFLOW and cytotoxicity using HALO-Tox HT

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Multiplexing OxyFLOW™ with HALO®-96 Tox

Etoposide causes single- and double-stranded DNA breaks. The diagram to the left shows the result of treating normal human peripheral blood with etoposide in a dose-dependent manner. The mononuclear cells were incubated for 4 or 7 days with etoposide. A aliquot from each dose was used to determine the presence of oxidative DNA damage using OxyFLOW™. A second aliquot was cultured to determine the effect of etoposide on peripheral blood in vitro multipotential stem cells (CFC-GEMM). A dimethylsulphoxide (DMSO) vehicle was used to dissolve the test compound. The results from OxyFLOW™ demonstrate that DMSO itself, can produce a significant toxic effect on the cells. When the dose of etoposide increases, there is an increase in the fluorescence intensity up to 1µM. This corresponds to the levels of intracellular ATP seen up to 1µM. Doses greater than 1µM cause a decrease in fluorescence intensity that correlates with the decrease in intracellular ATP levels indicating cytotoxicity. At the highest dose (0.1mM) of etoposide, few cells remain to be detected using OxyFLOW® and this again correlates with the very low intracellular ATP levels found using HALO®. This is not only an excellent example of assay multiplexing, but clearly demonstrates that HALO®-96 Tox can be used as a cytotoxic assay and that the mechanism of action of etoposide can be elucidated with OxyFLOW™. 


Basic Cell Preparation Procedures for In Vitro Culture
  • Initial preparation of organs and tissues for dissociation
  • Dissociation of organs and tissues into single cell suspensions
  • Cell washing
  • Viability by dye exclusion methods (e.g. 7-AAD and flow cytometry)
  • Viability by metabolic methods (LIVEGlo™)
  • Total nucleated cell count
  • Fractionation by density gradient centrifugation
  • Fractionation by Percoll
  • Fractionation over serum
  • Fractionation by adherence
  • Cell separation and purification by magnetic beads
  • Mononuclear cell count

In Vitro Cell Maintenance or Expansion Procedures
  • Cell expansion on tissue culture-treated surfaces
  • Cell expansion on adherent foils
  • Cell expansion on non-adherent foils
  • Cell expansion in bags
  • Cell expansion on feeder layers
  • 3D culture and cell expansion
  • Cell expansion on collagen-treated surfaces
  • Cell expansion on matrix-treated surfaces
  • Removal and purification of cells
  • Cell counts
  • Cell viability

 

Culture Conditions

Virtually all cells have to be cultured in a fully humidified atmosphere containing CO2, usually at 5%. It is not often that a company offers culture conditions under low oxygen tension. In fact, culturing cells under low oxygen tension helps reduce oxygen toxicity, keeps molecules in a reduced state and improves plating efficiency and therefore assay sensitivity. At HemoGenix® all cultures involving lympho-hematopoietic cells are performed under low oxygen tension approx. equivalent to venous oxygen tension (35mm Hg or 5% O2). Mesenchymal stem cells are also best cultured under low oxygen tension. Some organs, however, exhibit wide ranges of oxygen tension. These include the liver and kidney. As a result, contract services using cells from these and other organs may be offered using atmospheric and low oxygen tension.