Target identification, validation & discovery

A. Target identification/discovery is the first stage of drug development process. The key objective is to identify a novel or a putative therapeutic target for a disease with unmet medical need. Often proteins including enzymes, cell signalling receptors, structural proteins and regulatory factors are the most common targets. However, nucleic acids such as mRNA, carbohydrates and lipids are increasingly being recognised as therapeutic targets.

Target identification can be based on:
  • a published (peer-reviewed) data demonstrating a link between the target and a disease of interest: literature target, patents etc.
  • ‘Omic’ studies: Transcriptional profiling and proteomics data that leads to the discovery of genes/proteins with differential expression patterns in diseased states
  • Evidence of a novel biology that regulates the disease pathway or the target of interest.
B. Target validation includes the following stages:
  • Discovering a biomolecule of interest
  • Investigating the expression of the molecular target within the tissue of interest
  • Evaluating its potential as a target to understand if it is directly involved in the disease process
  • Designing a bioassay to measure biological activity to verify if target modulation produces the desired therapeutic effect.
  • Constructing a high-throughput screen.
  • Performing screening to find hits and evaluating the hits

We offer a range of strategies and capabilities for modulating gene expression in vitro.

These include but are not limited to the use of antibodies, negative dominant controls, antisense oligonucleotides, ribozymes, and small-interfering RNAs.

Our services to support target validation and discovery:
  • We can offer support and assay design for protein and novel target expression in a range of normal and diseased tissue, including:
    • - IHC and novel marker assay development & co-localisation studies
    • - phosphorylated marker assay development
  • Low-throughput target validation assay that represents biology
  • Molecular level
    • - Screen enzyme inhibitors or activators
  • Cellular Level
    • - Verify the involvement of the protein in the disease state (often use gene silencing siRNAs).
    • - Understand the protein pathways and interactions.

Cell viability and apoptosis/cytotoxicity

a. Green Cyanine dye assay - The dye used for this Cytotoxicity Assay is a trademarked asymmetric green cyanine dye. The assay is based on the principles of measuring the membrane integrity that occur as a result of cell death. The dye is excluded from viable cells but specifically binds only to the DNA realised from the dead cells. Binding of the dye to the DNA changes its property and makes it fluorescence. Since viable cells do not release any DNA there is no fluorescence observed in these cells. Therefore, the fluorescence signal produced by the binding interaction with dead cell DNA is proportional to cytotoxicity.

b. Alamar Blue assays - designed to measure quantitatively the proliferation of various human and animal cell lines, bacteria and fungi. The alamarBlue® Assay incorporates a fluorometric/colorimetric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth.

c. LDH Cytotoxicity Assay - Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Plasma membrane damage releases LDH into the cell culture media. Extracellular LDH in the media can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan product that can be measured at 490nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity.

d. MTT assay - This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (eg. isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.


Cell migration/chemotaxis; Scratch wound migration (Chemokinesis)

Cell migration is a characteristic feature of malignancy and inflammation. It is one of the key processes involved in angiogenesis and is therefore linked to tumour metastasis. Cell migration is induced by various agents including growth factors, chemokines and involves a complex machinery of cell signaling pathways. These pathways therefore present an attractive target for the management of inflammatory diseases or tumour development. Cell migration/scratch wound migration assays can be applied to investigate the effects of potential inhibitors using transwell migration assays or scratch wound chemokinetic assays.

For cell migration experiments, cells of interest are added to the top of transwell inserts in a 24-well notched plate in the presence or absence of inhibitors. The chemoattractant is added to the bottom chamber and incubated for up to 4 hrs at 37⁰ C. Following the incubation, cells present in the bottom chamber are quantified using a haemocytometer or fluorescently stained and quantified. IC50 values are then generated.

For the scratch wound migration experiments, cells of interest are plated onto 24-well plates in relevant culture medium till confluent. This is followed by mechanical scratching of the confluent cell layer to mimic wounding of cells and incubating at 37⁰C. The cells may then migrate for 24 hours in the presence or absence of inhibitors. Following this the cells are stained, photographed and the fluorescence quantified for generation of IC50 values.


Angiogenesis assay

The angiogenic process:

1- Permeability Assays:

a. To measure the ability of compounds to reduce cell-cell adhesion (transendothelisation) or cell-extracellular matrix adhesion (GAP junctions, tight junctions, etc.)

FITC-dextran assay: Cells are seeded in the top chamber at full confluence and exposed to test compounds, together with FITC-dextran. Interruption of the cell layer allows FITC-dextran to fall into the bottom chamber.


b. TransEpithelial Electrical Resistance (TEER):

Measures the conduction of electricity in a layer of cells. The conduction is different if the cell layer is continued or discontinued.

2- Proliferation and apoptosis
  • Seed the cells in 96 wells or coverslips.
  • Treatments with compounds, siRNA, antibodies, etc, in presence of substrates when required.
  • If necessary, apposite time and density curves will be performed.
  • Stop the reactions and/or fix cells, determine outcome by luminescence, colorimetric, cell counting.
  • Analyse percentage of alive/dead cells versus positive and negative controls.

BrdU, MTT, Alamar Blue, Ki-67, Caspase3/7, TUNEL assay

To quantify the number of alive and/or dead cells as a result of treatments

3- Matrigel network formation
to determine the ability of cells to form connections, follow chemoattractive signals, secrete signals.
  • Cells are seeded in a layer of Matrigel (with growth factors or reduced)
  • Incubated at 37°C in presence of activators/inhibitors – blocking antibodies, siRNA, growth factors, etc.
  • Remove media and fix with PFA (optional)
  • Image analysis
4- Gene expression QuantiGene Affymetrix expression, Real Time PCR, end point PCR

5- Cytokines/proteins expression Luminex protein expression, ELISA, Western Blot, In-cell Western Blot, In-cell ELISA.

Biomarkers

Angiogenesis Cancer Adhesion/Invasion
Amgiopoietin-2 Amgiopoietin-2 MMP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13
BMP-9 sCD40L proMMP9
EGF EGF TIMP 1,2,3,4
Endoglin Endoglin Collagen IV
EGF-1 and FGF-2 sFASL ICAM-1
Follistatin HB-EGF Galectin1, 3, 9
G-CSF IGFBP-1 Fibronectin
HB-EGF IL-6 VCAM
HGF IL-8  
  IL-18  
  IGF-α  

Signalling Pathway Activation Biomarkers

Luminex QuantiGene or Multiplex analytes(total and phosphorylated In-cell Western and In-cell ELISA (total and phosphorylated)
Erk/MAPK1/2 AKT
Akt P38 MAPK
STAT1, STAT3, STAT5A/B MPKK
JNK/SAPK1, c-JUN HSP27
p70 S6 Kinase JNK and c-JUN
NFkB P53
Caspase PARK
GSK caspase


6- Migration and Invasion assays

a. Scratch assay:
Monolayer of cells is scratched to form a gap and treated with an anti-proliferative agent and test compound - outcome: the gap closure compared to controls.

b. Boyden chamber:
Cells seeded at the top chamber and chemoattractant or different cell line in the bottom chamber – outcome: number of cells migrated to the bottom chamber, compared to controls.

c. Matrigel invasion:
Similarly to the Boyden chamber, cells are seeded on top of Matrigel and their activity to degrade the Matrigel is measured on the migrated cells.


7- Shedding assays:
  • The process of releasing cell surface components, alone or into vesicles. High levels of shedding activity are associated with cancer.
  • Shedding process activates: adhesion molecules, growth factors, cytokines precursors, receptors, enzymes.
  • Junction proteins (tight junctions, desmosomes, adherens): Claudin, JAM-A, Occludin, Cadherin, Desmoglein, etc.


Vascular permeability Assay

What is Vascular Permeability?

Vascular permeability is the permeability of the vessels carrying blood. The endothelial cells lining the vessels pay a major role in vascular permeability, as it defines the semi-permeable barrier between blood and the interstitial spaces. The permeability through the endothelial layer takes place via the vesicular transport and the paracellular transport pathways. These pathways are composed of several protein structural components, such as adherens, tight, and gap junction complexes, and desmosomes. The sub-structural components such as connexins, integrins, cadherins, catenins, occludins, desmoplakins, selectins, and platelet endothelial cell adhesion molecule-1 (PECAM-1) regulate the permeability.

Blood carries nutrients and waste from one part of the body to another. Therefore, the permeability of the blood vessels is important to maintain the homeostasis of the body. Any irregularity in the vascular permeability leads to acute inflammation and pathologies associated with angiogenesis such as tumours, wounds, and chronic inflammatory diseases.

Disruptions of the barrier integrity are manifested as microvascular hyperpermeability, which is associated with many systemic disease states. The barrier integrity for normal venule, acute vascular hyperpermeability (AVH) and chronic vascular permeability (CVH) are shown in Figure 1. Pathological angiogenic disease states include heart disease, diabetes, cancer, stroke, hypertension, arthritis, and Alzheimer’s. Increases in tissue permeability may be caused by weak, haemorrhaging vessels that become oedematous, and intensifies with irregular fluid flow through the vessels. Expanding the knowledge of endothelial junction behaviour and the agents that influence that behaviour will lead to new therapies for controlling endothelial permeability.

Normal venule, Acute Vascular Hyperpermeability (AVH), Chronic Vascular Hyperpermeability (CVH)

Figure 1: The barrier integrity of Normal Venule, AVH and CVH.

The vesicular transport and the paracellular transport pathways can be clearly seen.

Different Assays to measure Vascular Permeability:

1. Trans-endothelial epithelial resistance (TEER) Measurement:
Principle:

Transepithelial/transendothelial electrical resistance (TEER) is the measurement of electrical resistance across a cellular monolayer and is a very sensitive and reliable method to confirm the integrity and permeability of the monolayer. Although the measurement of TEER and of transepithelial passage of marker molecules are both indicators of the integrity of the tight junctions and of the cell monolayer, they determine different experimental parameters. TEER reflects the ionic conductance of the paracellular pathway in the epithelial monolayer, whereas the flux of nonelectrolyte tracers (expressed as permeability coefficient) indicates the paracellular water flow, as well as the pore size of the tight junctions. The advantages and wide use of the TEER method is because it is non-invasive and can be applied to monitor live cells during their various stages of growth and differentiation.

One method of measuring TEER is the Ohm's Law, where the electrical resistance of a cellular monolayer is measured in ohms, which is a quantitative measure of the barrier integrity.

Protocol:

The classical setup for measurement of TEER, as shown in Figure 2, consists of a cellular monolayer cultured on a semipermeable filter insert which defines a partition for apical (or upper) and basolateral (or lower) compartments. For electrical measurements, two electrodes are used, with one electrode placed in the upper compartment and the other in the lower compartment and the electrodes are separated by the cellular monolayer.

Figure 2: 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.


Result:
Cell type used in In vitro Model TEER (Ω.cm2) Equipment Used Ref
Blood Brain Barrier
Immortalized Human Brain Endothelial Cell Line (hCMEC/D3) 100 EVOM/Chopstick Daniels et al. 2013
GI models
Caco-2 1400 - 2400 EVOM/Chopstick Hilgendorf et al. 2000
Caco-2 and HT29-MTX 110 - 185 EVOM/Chopstick Hilgendorf et al. 2000
Alveolar Epithelia

 

Human Alveolar type-II Epithelial cells (HAEpC) 1000 - 2000 EVOM Fuchs et al. 2003
A549 45 - 100 EVOM/STX2 Barar et al. 2005
Human pulmonary microvascular endothelial cells (HPMEC) and NCI-
H441
565 ± 48 EVOM/STX2 Hermanns et al 2004
A549, macrophage like cells (THP-1), mast cells (HMC-1), endothelial cells (EA.hy 929) 250 EVOM/STX2 Daniels et al. 2013
Primary human type-II alveolar epithelial cells (HAT-II) and HPMEC 1730 ± 460 EVOM/STX2 Hermanns et al 2009
Airway Epithelia
Human Bronchial Epithelial cell line (Calu-3) 300 - 600 EVOM/STX2 Foster et al. 2000
Human Bronchial Epithelial cell line (Calu-3) 1126 ± 222 EVOM/STX2 Mathia et al. 2002
Human Bronchial Epithelial cell line (Calu-3) 50-60 
(in microwells)
EVOM/STX2 Bol et al. 2014
Calu-3, MRC5 and dendritic cells 200 EVOM/STX2 Harrington et al. 2014
Diseased bronchial epithelial cell line (CFBE41o-) 250 EVOM/STX2 Ehrhardt et al. 2006
References:

Barar J. Modulation of Cellular Transport Characteristics of the Human Lung Alveolar Epithelia. Iranian Journal of Pharmaceutical Research. 2005; 3:163–171.

Bol L, Galas JC, Hillaireau H, et al. A Microdevice for Parallelized Pulmonary Permeability Studies. Biomed Microdevices. 2014; 16:277–85.

Daniels BP, Cruz-Orengo L, Pasieka TJ, et al. Immortalized Human Cerebral Microvascular Endothelial Cells Maintain the Properties of Primary Cells in an in Vitro Model of Immune Migration Across the Blood Brain Barrier. J Neurosci Methods. 2013; 212:173–9.

Ehrhardt C, Collnot EM, Baldes C, et al. Towards an in Vitro Model of Cystic Fibrosis Small Airway Epithelium: Characterisation of the Human Bronchial Epithelial Cell Line CFBE41o. Cell Tissue Res. 2006; 323:405–415.

Foster KA, Avery ML, Yazdanian M, et al. Characterization of the Calu-3 Cell Line as a Tool to Screen Pulmonary Drug Delivery. Int J Pharm. 2000; 208:1–11.

Fuchs S, Hollins AJ, Laue M, et al. Differentiation of Human Alveolar Epithelial Cells in Primary Culture: Morphological Characterization and Synthesis of Caveolin-1 and Surfactant Protein-C. Cell Tissue Res. 2003; 311:31–45.

Harrington H, Cato P, Salazar F, et al. Immunocompetent 3D Model of Human Upper Airway for Disease Modeling and In Vitro Drug Evaluation. Mol Pharm. 2014

Hermanns MI, Fuchs S, Bock M, et al. Primary Human Coculture Model of Alveolo-Capillary Unit to Study Mechanisms of Iinjury to Peripheral Lung. Cell Tissue Res. 2009; 336:91–105.

Hermanns MI, Unger RE, Kehe K, et al. Lung Epithelial Cell Lines in Coculture with Human Pulmonary Microvascular Endothelial Cells: Development of an Alveolo-Capillary Barrier in Vitro. Lab Invest. 2004; 84:736–52.

Hilgendorf C, Spahn-Langguth H, Regardh CG, et al. Caco-2 Versus Caco-2/HT29-MTX CoCultured Cell Lines: Permeabilities Via Diffusion, Inside- and Outside-Directed Carrier-Mediated Transport. J Pharm Sci. 2000; 89:63–75.

Mathia NR, Timoszyk J, Stetsko PI, et al. Permeability Characteristics of Calu-3 Human Bronchial Epithelial Cells: In Vitro-in Vivo Correlation to Predict Lung Absorption in Rats. J Drug Target. 2002; 10:31–40.



2. Vascular Permeability Determination:
Principle:

This Assay is performed in a 24-well receiver plate with 24 individual hanging cell culture inserts.

The inserts contain 1 μm pores within a transparent polyethylene terephthalate (PET) membrane. Each insert has been pre-coated with an optimized concentration of type I rat-tail collagen. The high pore density membranes permit apical and basolateral access of cells to media and permeability molecules of interest.

Within this In Vitro Vascular Permeability Assay, endothelial cells are seeded onto the collagencoated inserts. An endothelial monolayer form in several days, which occludes the membrane pores. The cell monolayer is then treated with cytokines, growth factors, or other compounds of interest. After treatment, a high molecular weight FITC-Dextran is added on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer at a rate proportional to the monolayer’s permeability. The extent of permeability can be determined by measuring the fluorescence of the receiver plate well solution. This Assay is ideal for measuring compounds that may disrupt or protect an endothelial monolayer.

Protocol: Figure 3: Representation of the vascular permeability assay using the FITC Dextran.

Result:
Figure 4. Example staining and permeability analysis of HUVEC monolayers. .

HUVEC at passage 4 were seeded at 200,000 cells per insert and cultured for 72 hours in growth medium ("No Monolayer" negative control cultured in growth medium only). Following this culture period, monolayers underwent “No Treatment” (growth medium only) or treatment with 100 ng/mL TNF-α in growth medium for 23 hours. Monolayer staining and FITC-Dextran permeability testing were performed as described in the Assay Protocol. Stained cells were brightfield-imaged on an inverted microscope at 5X objective magnification. Fluorescence intensities were quantified. The “No Monolayer” sample demonstrated high permeability in the absence of an occlusive endothelial cell monolayer.

The "No Treatment" sample exhibited a visually confluent monolayer, as supported by the finding of low FITC-Dextran permeability (a “positive control” for monolayer integrity). Disruption of monolayer integrity was observable both visually and by quantification of increased plate well solution fluorescence following TNF-α treatment.

Figure 5. Time-courses for stimulation of vascular permeability by IL-1b and TNF-a.

HUVEC were seeded at 200,000 (for IL-1β, passage 4) or 100,000 (for TNF-α, passage 3) cells per insert and cultured for 72 hours in growth medium. Following this culture period, monolayers underwent “No Treatment” (growth medium only) or treatment with 100ng/mL IL-1β or TNF-α in growth medium for a range of durations. FITC-Dextran permeability testing was performed as described in Figure 3. Duplicate samples demonstrated low permeability for non-treated samples, and time-dependent increases in permeability following IL-1β or TNF-α treatment. Bars are mean +SEM.



3. Tight Junction Protein Expression:
Principle:

Endothelial cells provide a non-thrombogenic monolayer surface that lines the lumen of blood vessels and functions as a cellular interface between blood and tissue. Epithelial cells line provides a protective layer for both the outside and the inside cavities and lumen of the body. Epithelial and endothelial cells are connected to each other via intercellular junctions that differ in their morphological appearance, composition and function (See Figure 6). The tight junction or zona occludens is the intercellular junction that regulates diffusion and allows both of these cell layers to form selectively permeable cellular barriers that separate apical (luminal) and basolateral (abluminal) sides in the body, thereby controlling the transport processes to maintain homeostasis.

The In Vitro Vascular Permeability Imaging Assay provides the reagents necessary for affixing a thin, uniform layer of biotinylated gelatin to a glass culture substrate which, in the presence of an endothelial monolayer, binds to a fluorescently labelled streptavidin only at sites of intercellular permeability. A poly-L-lysine coating is first adsorbed to the glass substratum. The substrate is then treated with a dilute glutaraldehyde solution to bi-functionally “activate” the surface for further protein binding. Subsequent incubation of the surface with biotinylated gelatin allows covalent coupling between the poly-L-lysine and gelatin via reactive aldehyde (-CHO) groups. The biotincoated glass is now prepared for cell culture by disinfection with 70% ethanol, followed by quenching of free aldehydes with amino acid-containing growth medium.

The endothelial cell of interest is seeded onto the gelatin surface and allowed to form a confluent monolayer. Treatment compounds of interest may be introduced at desired time points during the culture period. Sites of intercellular permeability are then stained with fluorescent streptavidin, which are microscopically visualized and may be quantified using image analysis software algorithms. The assay also provides anti-VE-cadherin and DAPI, for visualization of adherens junctions and nuclei, respectively, to allow colocalization of sites of adherens junction re-modeling and increased permeability. The basic method allows potential activators or inhibitors to be investigated for their influence on the degree and sites of vascular permeability. The assay may be further combined with immunocytochemical staining for other molecules of interest to colocalize sites of permeability with signalling events.

Immunostaining for proteins characteristic of tight junctions (occluding, ZO-1 and ZO-2) can provide qualitative insights into the barrier integrity of an endothelial or epithelial monolayer. This assay may be used for assessing activity of inhibitors and promoters of vascular permeability, and correlating permeability with ultrastructural features and signalling events as seen in Figure 7.

Figure 6. Structural organization of endothelial cell intercellular and matrix interactions.

Interenthothelial junctions (IEJs) comprised of tight junctions, adherens junctions, and gap junctions interact through actin cytoskeleton with integrin receptors enabling endothelial cell adhesion with contiguous cells and to the underlying matrix. Occludin, claudins, and junctional adhesion molecules (JAMs) are the backbones of tight junctions, whereas vascular endothelial cadherin (VE-cadherin) is required for the formation of adherens junctions. Connexins form gap junctions. Intracellular domains provide junctional stability through their linkages with the actin cytoskeleton via catenins (α, β, γ, and p120) or zona occluden 1 protein (ZO). Gap junctions allow the rapid exchange of information between cells. The cytosolic domains of integrins are linked with actin cytoskeleton through proteins talin and vinculin (Vin), involved in integrin-mediated signalling. Membrane metalloproteases (MMPs) control remodelling of the extracellular matrix (ECM). FN: fibronectin; VN: vitronectin.

Agonists increase intracellular Ca2+, which, by activating the myosin light-chain kinase (MLCK) and RhoA-Rho kinase pathway, induces actin stress fibre formation, leading to disruption of AJs. Additionally, protein kinase C (PKC), Src, and end-binding protein 3 (EB3) disrupt AJs adhesion by either phosphorylating them or by increasing microtubule dynamics. Several mechanisms are activated in parallel or synergistically to induce re-annealing of AJs. For example, focal adhesion kinase (FAK) phosphorylates neural Wiskott–Aldrich syndrome protein (N-WASP), enabling actin related protein 2/3 (Arp2/3) to mediate cortical actin formation. Activated N-WASP also links p120-catenin to Arp2/3 and actin to stabilize AJs. FAK also induces Rac1 activity by inhibiting RhoA activity, which may further stabilize AJs. Activated Cdc42 may merge with FAK signalling to restore AJs formation.

Imaging and Analysis:
  • Perform fluorescent microscopic imaging of samples using appropriate fluorescence filters as listed in Table 2 below.
  • Selection of imaging magnification and/or modality (e.g., widefield or confocal fluorescence) will depend upon application of interest. Lower magnifications (e.g., 20X objective) may be sufficient for quantification of large scale changes in permeability, while co-localization studies may require higher magnification/resolution visualization (e.g., 63X oil objective). Optimal imaging settings/techniques must be determined by the end user.

Result:
Figure 8. Imaging of the effects of thrombin and forskolin on permeability of HUVEC monolayers.

Thrombin disrupts VE-cadherin-containing tight junctions and increases permeability of the monolayer to fluoresceinstreptavidin. In contrast, forskolin increases tight junctions containing VE-cadherin (red) and reduces penetration of fluorescein-streptavidin (green) through the monolayer. The nucleus was stained with DAPI (Blue). Figure A and B were imaged at 20X dry lens and 63X oil immersion objective lens.


4. Release of Inflammatory mediators:
Principle:

A multitude of vasoactive cytokines, growth factors, and signal modulators react with endothelial cell sub-structural components to control permeability. Vascular endothelial growth factor (VEGF), interleukin-1 alpha and beta (IL-1α and IL-1 β), tumour necrosis factor-alpha (TNF-α), and interferon gamma (IFN-γ) have been shown to increase endothelial monolayer permeability.

Protocol:

The following panels of cytokines, chemokine, growth factors and inflammation markers are multiplexed on Luminex MAGPix platform.

Cytokines, chemokine and growth factors:

BDNF IL-6 IP-10
EGF IL-7 LIF
Eotaxin IL-8 MCP-1
FGF-basic IL-9 MIP-1α MIP-
GM-CSF IL-10
GROα HGF IL-12 p70 NGFβ
IFNγ IL-13 PDGF-BB 
IFNα IL-15 PLGF
IL-1RA IL-17A RANTES SCF
IL-1β IL-18 SDF1α
IL-1α IL-21 TNFα
IL-2 IL-22 TNFβ
IL-4 IL-23 VEGF-A
IL-5 IL-27 VEGF-D
  IL-31  

Inflammation:

E-Selectin IL-4 IP-10
GM-CSF ICAM-1 IL-6 MCP-1
IFNα IL-8 MIP-1α
IFNγ IL-10 MIP-1β
IL-1α IL-12p70 P-Selectin
IL-1β IL-13 TNFα
  IL-17A  
Cell Models Available:

The successful application of a system to predict drug absorption depends on how closely the in vitro model can mimic the characteristics of the in vivo barrier integrity. These in vitro models can be based on primary cells or immortalized cell lines.


Some of the Immortalized Cell lines are as shown below:

Cell Invasion Assay

Cell invasion is a process whereby the cells have the ability to migrate from one location to another through an extracellular matrix (ECM) or basement membrane extract (BME). Cell invasion involves binding of cells to the ECM components (e.g. type II collagen, fibronectin and laminin) through integrin and/or non-integrin receptors followed by degradation/manipulation of ECM using enzymes [such as matrix metalloproteases (MMPs) and urokinase type plasminogen activator (uPA)]. Both normal and malignant cells display the property of invasion during inflammation and metastasis respectively. Thus, understanding the mechanism of cell invasion is important for various biological processes.

Cell invasion assays can be used to study the effect of the drugs on inflammation or metastasis. These assays can be performed in 24 or 96 plate colorimetric format. The cells are fluorescently stained and plated onto membrane inserts. The membrane inserts can be coated with common barriers such as collagen, laminin, fibronectin or more complex ECM and BME. The chemoattractant is added to the bottom chamber and incubated at 37⁰ C for the required time. The plates are then read on florescent plate reader after incubation.


Membrane Integrity Assay

This assay estimates the number of nonviable cells present in multiwell plates. Cell viability is most often defined based on cell membrane integrity and is most commonly measured by observing the exclusion of Trypan blue or other vital dyes. Measuring leakage of components from the cytoplasm into the surrounding culture medium has been widely accepted as a valid method to estimate the number of nonviable cells. This assay measures the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium is measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into resorufin. Reagent mix does not damage healthy cells; therefore, researchers can measure released LDH directly in assay wells containing a mixed population of viable and damaged cells (Courtesy: Promega).


Cell Proliferation/viability/cytotoxicity

One of the most commonly applied applications of Proliferation assay is to test the effects of anti-cancer compounds on malignant cells either grown as monolayers in cell culture plates or in cell culture suspensions. This 2D assay can be used to screen large number of compounds reliably and efficiently.

Cell Proliferation Assay kit consists of a green fluorescent nucleic acid stain and a background suppression dye. The nucleic acid dye is a live cell-permeable reagent that stains the nucleus of mammalian cells. The suppression dye is impermeable in live cells and suppresses “green” fluorescence. This assay is therefore based on both DNA content and membrane integrity. Fluorescence intensities are measured with a fluorescence microplate reader and is an accurate measurement of cytotoxicity and cell proliferation. These values are normalized to the control treatment and IC50 curves generated.


DNA damage and repair

Mechanisms underlying DNA damage and repair in cells exposed to exogenous agents such as ionizing radiation, ultraviolet light, oxidative stress and chemical mutagens can be assesses by the following assays:

8-oxo-dG assay
An immunoassay designed to detect and quantify 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in plasma, urine, saliva samples and from DNA extracted from cultured and adherent cells.
8-oxo-dG is a frequently used biomarker of oxidative DNA damage and oxidative stress. Reactive oxygen and nitrogen species generated by normal metabolic processes and a variety of environmental factors cause the hydroxylation of guanosine at the N7-C8 bond. 8-oxo-dG is unstable and, as a result, polymerases preferentially insert adenine opposite it. If the modified DNA is not repaired, the oxidative damaged adducts can lead to G-to-T transitions. Increased levels of 8-oxo-dG are associated with the aging process as well as with a number of pathological conditions including cancer, diabetes, and hypertension (Courtesy: R & D Systems)

Superoxide Dismutase Assay
To detect Superoxide Dismutase (SOD) activity in cell and tissue extracts. SODs are metalloenzymes that catalyze the dismutation of the superoxide radical (O2-) into hydrogen peroxide (H2O2) and molecular oxygen (O2), providing an important defense against oxidative damage. In the Superoxide Dismutase Assays, superoxide ions are generated from the conversion of xanthine and O2 to uric acid and H2O2 by Xanthine Oxidase (XOD). The superoxide anion then coverts a tetrazolium salt into a formazan dye. Addition of SOD to this reaction reduces superoxide ion levels, thereby lowering the rate of formazan dye formation. SOD activity in the experimental sample is measured as the percent inhibition of the rate of formazan dye formation.
SOD Inhibition Assay Mechanism XOD and SOD Antagonism in the Generation of Formazan Dye.
The conversion of xanthine and O2 to uric acid and H2O2by XOD generates superoxide radicals. The superoxide anions reduce a tetrazolium salt (nitroblue tetrazolium [NBT] or WST-1) to a colored formazan product (NBT-diformazan or WST-1 formazan) that absorbs light. SOD scavenges superoxide anions, thereby reducing the rate of formazan dye formation (Courtesy: R & D Systems)

PARP1 Enzyme Activity Assay
An assay for quantifying PARP1 & PARP2 activity. This employs nicotinamidase to measure nicotinamide generated upon cleavage of NAD+ during PARP-mediated poly-ADP-ribosylation of a substrate, thereby providing a direct, positive signal assessment of the activity of PARP1 & PARP2.
To perform the assay, a PARP enzyme, β-NAD, activated DNA, test compound, and recombinant nicotinamidase enzyme are combined and incubated for 30 minutes. During the incubation, the activated DNA triggers PARP1 or PARP2 to produce poly(ADP-ribose) and nicotinamide. In a secondary reaction, the nicotinamidase enzyme converts the nicotinamide into nicotinic acid and NH3+ (free ammonia). To generate a signal for readout, a proprietary developer reagent is added and the signal is read using a fluorescent plate reader. The robust performance of this assay makes it appropriate for measuring PARP1 and PARP2 activity as well as for screening of activators and inhibitors of PARP enzymes (Courtesy: Millipore).


Collagen gel contraction

Objective: To assess contractile properties of human airway smooth muscle cells (HASM)

  • Collagen gels impregnated with HASM cells are treated in the presence/absence of agonist/antagonist
  • gels are photographed at specific time points and the gel size is measured and quantified as an indicator of airway smooth muscle contraction


Measurement of Mucin production by immunoassays

  • 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


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-1α, IL-6, IL-8, TNFα, TGFβ
  • Other Endpoints: Oxidative Stress, Apoptosis


Mast cell degranulation assays

  • HBEC/BEAS 2B and HMC-1 Co-culture model
  • Sensitisation using either 2.5 µg/ml human IgE or Calcium Ionophore to induce mast cell degranulation
  • Measure 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


B lymphocytes IgE release assay

  • Stimulation of B lymphocyte cell line with IL4, IL5 and IL13
  • Measure IgE release by immunoassays


Immunogenic Cell Death Assays

  • CAL (calreticulin) receptor expression by Immunofluorescence/histology, immunoassays
  • Secretion ATP –luminescence assays
  • Release of intracellular HMBG1 measured by immunoassays, localisation of intracellular HMGB1 by flow cytometry
  • HSP70 release measured by immunoassays, western blot
  • HSP 90 release measured by immunoassays, western blot
  • induction of a type-1 interferon response – detection of IFNα1, α2 and β1 by RT-PCR
    • type 1 interferon response includes STAT3 activation by Tyr phosphorylation and tyrosine kinase activation (mainly, but there are more targets) – measure pSTAT3 and pTYK as one of the pathway activation biomarkers suggesting induction of type-1 interferon response.


Monocyte Recruitment and Adherence:

  • Monocytes are the first immune cells to be recruited to the site of inflammation and injury. The monocytes need to adhere to the site of injury to differentiate and activate
  • Monocytes from normal/unhealthy patients are isolated and co-cultured to monolayers of HUVEC cells.
  • The number of monocytes recruited and adhered are counted after Coomassie/Giemsa staining.
  • End point:
    • Qualitative data: Images
    • Quantitative data: Percentage of monocyte adhesion.


Cell apoptosis

Apoptosis is the cellular process of programmed cell death. It plays a critical role in development and immunity, as well as cancer and neurodegenerative disease. Annexin V/Propidium Iodide staining is used to distinguish between apoptotic, necrotic and dead cells. Apoptotic cells stain positively for Annexin V that binds to phosphotidylserine (PS) following their translocation to the extracellular surface of the cell membrane. These cells are negative for Propidium Iodide. Dead cells stain positive for both Annexin V and Propidium Iodide and viable cells are negative for Annexin V/Propidium Iodide staining.


In Vitro Vascular Permeability assessment

Vascular endothelial cell lining acts as a semi-permeable barrier between blood and the interstitial spaces of the body. This is composed of desmosomes, intercellular adherens, tight and gap junction complexes. Junction substructure components such as connexins, integrins, cadherins, catenins, occludins, desmoplakins, selectins, and platelet endothelial cell adhesion molecule-1 (PECAM-1) regulate permeability of ions, nutrients, therapeutic agents, and macromolecules. Disruptions of the barrier integrity are manifested as microvascular hyperpermeability, which is associated with many systemic disease states.
Test Principle: In Vitro Vascular Permeability Assay is performed in a 24-well receiver plate with 24 individual hanging cell culture inserts. The inserts contain 1 µm pores within a transparent polyethylene terephthalate (PET) membrane. Endothelial cells are seeded onto the collagen-coated inserts. An endothelial monolayer forms in several days, which occludes the membrane pores. The cell monolayer is then treated with cytokines, growth factors, or other compounds of interest. After treatment, a high molecular weight FITC-Dextran is added on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer at a rate proportional to the monolayer’s permeability. The extent of permeability an be determined by measuring the fluorescence of the receiver plate well solution (Courtesy: Millipore).


In-Cell ELISA/In-Cell Western Assay

A simple and convenient method for quantification of intracellular protein levels in whole cells.
Features of the In-Cell ELISA Colorimetric Detection Kit:

  • Colorimetric—compatible with standard ELISA plate readers
  • Simple, fast assay format
  • Ability to perform multiplex experiments
  • No special sample preparation required
  • Higher throughput compared with western blotting
  • Cost efficient (one well vs. one lane) compared to western blotting
While traditional western blot analysis is time consuming and only semi-quantitative, the in-cell ELISA is an accurate method to determine relative protein levels and degree of post-translational modification (PTM) among various cell types.

Applications
  • High throughput determination of relative protein expression levels
  • Monitor dose-dependent post-translational protein modification
  • Multiplex analysis of targets in multiple cell lines

The In-Cell ELISA Colorimetric Detection Kit enables the levels of target proteins to be compared in different wells. The relative amount of protein is determined using target-specific primary antibodies and a horseradish peroxidase (HRP)-conjugated detection reagent. Following the colorimetric measurement of HRP activity, the provided whole cell stain, Janus Green, is used to determine cell number. After staining, the results are analyzed by normalizing the absorbance (HRP activity) values to cell number, which adjusts for the cell plating differences. (Courtesy: Thermo Fisher Scientific)


Cell Phosphorylation

Application is to measure activation of intracellular signaling pathways to understand the molecular mechanism underlying the disease process or to elicit the mode of action of a chemical compound.

Bead based multiplex assays are capable of providing accurate relative quantitation of both total and phosphorylated forms of signaling proteins, revealing connections and crosstalk within your pathways of interest.

Below are some examples of the intracellular pathway assays that we can perform:

Akt p38 ERK TGFβ STAT Src Family
mTOR MAPK NFκB JNK RTK p70S6K