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Immunotoxicity 

 

Immunotoxicity testing summary performed at HemoGenix

 

For all immunotoxicity testing services please contact HemoGenix® at info@hemogenix.com, or call (719) 264-6250. Our immunotoxicity services are also available though Science Exchange.

 

 Step 1: Primitive Lympho-Hematopoietic Stem Cells and Lymphopoietic Progenitor Cells

The lymphopoietic and hematopoietic systems are joined at a very primitive stage of blood stem cell development. One of the cells that is common to both lineages is the SC-HPP or Stem Cell - High Proliferative Potential. This cells is quiescent, but can be affected by small molecules, which take effect once the SC-HPP is called upon to proliferate. Also affected would be the lymphopoietic progenitor cells, namely the P-Tcell and P-Bcell. 

 

Toxicity to these very primitive and early cells can be predicted using the HALO®-Tox HT and/or HALO® Real Time Platforms, but may also include colony-forming cell (CFC) assays.

  

 Step 2: Immunotoxicity Testing

In vitro immunotoxicity testing proper, usually begins once the T- and B-cells have been determined and demonstrate the functional characteristics of immune cells. This stage of immunotoxicity testing includes a number of functional tests that include, but not limited to, the following:

  • Lymphocyte proliferation/cytotoxicity assays 
  • Mixed lymphocyte culture/reaction (MLC/MLR)
  • Regulatory T-cell (Tregs) response
  • Cytotoxic T-cell (CTL) response
  • Antibody-dependent cell-mediated cytotoxicity (ADCC)
  • Antibody-drug congugates (ADC)
  • Immunophenotyping
  • Macrophage (M1/M2) response
  • Phagocytosis, oxidative burst (neutrophils), and migration
  • NK activity 
  • Dendritic cell maturation and co-stimulation
  • Cytokine/chemokine production and release
  • Cell injury and cell death
  • Extracellular markers of activation

 

The assays above can be performed using:

  • Peripheral blood mononuclear cells (PBMC)
  • Bone marrow (BM)
  • Purified lymphocyte cell populations

 

Species include:

  • Human
  • Primate
  • Dog
  • Rat
  • Mouse
  • Not all of these species may be available for particular studies

 ImmunoGlo™ Assays for Immunotoxicity Testing  
  • ImmunoGlo™-Tox HT is a lytic, high throughput, ATP bioluminescence platform used for screening compounds. It uses the same standardized readout as HALO®-Tox HT, but different lymphocyte culture conditions.
  • ImmunoGlo™ Real Time is a non-lytic, bioluminescence real time readout to determine the onset of immunotoxicity in a dose-dependent manner and can be simultaeously multiplexed with flow cytometric phenotyic analysis to determine which cell populations are affected by the insult.
  • ImmunoGlo™ MLC is a 1- or 2-way mixed lymphocyte reaction (MLR) or culture (MLC) in which the stimulator cells can be treated with mitomycin-C (as opposed to radiation) to inhibit their proliferation in a 1-way MLC.
  • ImmunoGlo™ TCP are assays in which co-stimulators, such as CD3 and CD28, are used to stimulate, detect and predict immunotoxicity.

 Immunotoxicity Testing and Mechanism of Action Panel


Immunotoxicity Testing and Mechanism of Action (MOA) Panel 

Assay Name Assay Type Pathway Readout
ImmunoGlo™-Tox HT Standardized iATP

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
ImmunoGlo™ TCP Standardized iATP

T-cell proliferation with

co-stimulators

(Tier 2)

Bioluminescence
ImmunoGlo™-Tox Real Time (RT) Reduction potential

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
ImmunoGlo™-MLC Standardized iATP

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
Cell cycle DNA marker Proliferation (Tier 2) Fluorescence
FlowDiff™ Immune membrane expression markers (e.g. CD3, 4, 8, 19, 73) Characterization / Differentiation (Tier 2) Fluorescence
LIVEGlo™ iATP Metabolic viability / Mitochondrial Function (Tier 1 & 2) Bioluminescence
Dye Exclusion Viability 7-Aminoactinomycin D (7-AAD) / Propidium Iodide (PI) Membrane integrity (Tier 1 & 2) Fluorescence
Mitochondrial ToxGlo™* ATP Mitochondrial dysfunction (Tier 2) Bioluminescence / Fluorescence
Glutathione Assay Glutathione Oxidative stress (Tier 2) Luminescence
OxyFLOW™ 8-Oxoguanine addudcts Oxidative DNA damage (Tier 2) Fluorescence
CaspaseGlo™* Apoptosis Caspases (Tier 2) Luminescence
Annexin-V / PI Apoptosis / Necrosis Phosphatidylcholine (Tier2) Fluorescence
GFkine™  Growth factor, cytokine production / release Multiple (Tier 2) Multiple

* Promega Corporation assays 

 

For more MOA studies, please navigate to the MOA page.