Pre-Clinical Laboratory Services
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:
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:
C. Cell-based in vitro assays:
To determine the efficacy of drug compounds, we offer a range of cell-based functional assays:
We have expertise in designing, developing, performing and analysing functional cell-based assays using different cell culture models including well-established cell lines and primary cells.
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 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.
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.
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
|Amgiopoietin-2||Amgiopoietin-2||MMP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13|
|EGF-1 and FGF-2||sFASL||ICAM-1|
|Follistatin||HB-EGF||Galectin1, 3, 9|
Signalling Pathway Activation Biomarkers
|Luminex QuantiGene or Multiplex analytes(total and phosphorylated||In-cell Western and In-cell ELISA (total and phosphorylated)|
|STAT1, STAT3, STAT5A/B||MPKK|
|p70 S6 Kinase||JNK and c-JUN|
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.
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.
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.
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.
|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|
|Caco-2||1400 - 2400||EVOM/Chopstick||Hilgendorf et al. 2000|
|Caco-2 and HT29-MTX||110 - 185||EVOM/Chopstick||Hilgendorf et al. 2000|
|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-
|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|
|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
|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|
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.
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.
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.
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.
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.
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.
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.
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:
|GROα HGF||IL-12 p70||NGFβ|
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.
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.
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).
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.
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:
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)
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).
Objective: To assess contractile properties of human airway smooth muscle cells (HASM)
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.
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).
A simple and convenient method for quantification of intracellular protein levels in whole cells.
Features of the In-Cell ELISA Colorimetric Detection Kit:
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: