are 3D models developed by Aspect Biosystems)
1. Target validation, qualification
- - Real time PCR
- - Gene silencing/knockdown assays
- - Western Blotting
- - Immunocytochemistry
- - MTT: indicator of mitochondrial metabolic activity
- - BrdU: detects 5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation
- - Green Cyanine dye assay: based on the principles of measuring the membrane integrity that occur as a result of cell death
- - Alamar Blue assay: designed to measure cell proliferation quantitatively by incorporating an oxidation-reduction (REDOX) indicator
- - LDH Cytotoxicity assay: measures release of a cytosolic enzyme, lactate dehydrogenase, from dead cells
- - MTT assay: indicator of mitochondrial metabolic activity
- - Caspase3/7 assay: an assay to detect caspase 3/7 activity as an indicator of apoptosis
4. Migration and Invasion Assays
5. Boyden chamber
- - Cells seeded at the top chamber and chemoattractant or different cell line in the bottom chamber.
- - End point: Number of cells migrated to the bottom chamber, compared to controls
6. Collagen gel contraction
3D collagen matrix has also been used in the studies of integrin signaling, cell apoptosis and cytoskeleton reorganization. Since three-dimensional matrix adhesions differ in structure, localization, and function from two-dimensional adhesions; and therefore, three-dimensional cell-matrix interactions may be more relevant biologically
Objective: To assess cell contractivity in vitro and screen cell contraction mediators.
- Collagen gels impregnated with target cells are resuspended in relevant growth media
- After polymerization, collagen gels are then pre incubated in the presence or absence of different concentrations of test compound and the contractile stimulant.
- The collagen gels are then photographed at specified time points over a 3h period by a blinded observer and the gel size as a percentage of the well area is calculated at specific time points using a professional imaging software
- All gel conditions are performed at least in duplicate.
- The quantified gel size is used as an indicator of target cell contraction
Example of a representative image of a Collage Gel contraction assay
7. Oxidative stress/DNA damage
- - 8-oxo-dG immunoassay: biomarker of oxidative DNA damage and oxidative stress
- - Superoxide Dismutase Assay: a colorimetric assay to detect superoxide Dismutase (SOD) activity in cell and tissue extracts as an indicator of oxidative stress
8. TEER measurement: Trans-endothelial/trans-epithelial electrical resistance (TEER) is the measurement of electrical resistance across a cellular monolayer, and a very sensitive and reliable method to confirm the integrity and permeability of the monolayer.
End-point: Quantitative data - Ohm meter readings.
TEER measurement with chopstick electrodes.The total electrical resistance includes the ohmic resistance of the cell layer RTEER, the cell culture medium RM, the semipermeable membrane insert RI and the electrode medium interface REMI.
9. FITC-Dextran permeability assay: FITC-Dextran molecules leak through cell monolayers where the permeability is compromised.
Quantitative data: Determined by measuring the fluorescence of the receiver plate well solution.
Qualitative data: Staining of the monolayer after FITC-Dextran assay.
Representation of FITC-Dextran permeability assay.
10. Vascular Permeability assays (airway endothelial cells)/Membrane Integrity Assays (airway epithelial cells)
- - Tight junction protein expression (V-Cadherin, occludins and claudins) by immunofluorescence
11. Measurement of Mucin production
- - Human airway epithelial cells are differentiated using air-liquid interface (ALI) culture method to form mucociliated epithelial cells
- - Cells are treated in the presence or absence of the test compound and mucus production is then measured by immunoassays
12. Respiratory toxicity and irritation in vitro model
- - Using differentiated airway epithelial in vitro model characterised by pseudo- stratified epithelium with tight junction formation, numerous apical cilia and apical mucin production
- - Positive Controls: Bleomycin (Irritation); Triton X-100 (Cell Death)
- - Biochemical Endpoints: MTT, LDH
- - Gene and Protein Expression Endpoints: IL-1a, IL-6, IL-8, TNFa, TGFβ
- - Other Endpoints: Oxidative Stress, Apoptosis
13. Mast cell degranulation assays
- - HBEC/BEAS 2B and HMC-1 Co-culture model
- - Sensitisation using either 2.5 µug/ml human IgE or Calcium Ionophore to induce mast cell degranulation
- - Measure the mast cell tryptase activity using a spectrophotometric method as an indicator of mast cell degranulation
- - Measure the release of other mediators of allergy and inflammation including histamine, lipoxin A4 by immunoassays
- - Measure the release of β-hexosaminidase
14. B lymphocytes IgE release assay
- - Stimulation of B lymphocyte cell line with IL4, IL5 and IL13
- - Measure IgE release by immunoassays
15. Pathway activation biomarkers
- Measure phosphorylation of downstream signalling events
- Multiplexing immunoassays
- Western Blot
- In-Cell ELISA
- Disease Biomarkers
16. Disease Biomarkers
- Measure release of cytokines, chemokines, growth factors and inflammatory mediators (mRNA and protein)
- Multiplexing immunoassays