Introduction / Principle
Cellular monolayer healing is monitored by an automated microscopy machine: the IncuCyte (ESSEN BIOSCIENCE) (quantification of cellular confluence).
Protocol
Format: 24 wells or 96 wells
Carrying out the wound of the cell monolayer at confluence with a "WoundMaker" (ESSEN INSTRUMENT).
Monitoring
of cell monolayer healing at the wound level over time in the presence or
absence of molecules of interest and/or trophic factors.
WoundMaker 24 wells "Pin block" 96 wells
WoundMaker (ESSEN INSTRUMENT)
Monitoring of healing on HaCaT cells
Molecular screening on purified targets typically aims to (1) modify interactions between biological targets or (2) modify the activity of a biological target (e. g. enzyme).
1) Examples of interaction tests:
Nature of the test :
. Protein-protein interactions
. Protein-nucleic acid interactions
. Interactions between soluble proteins and ligands.
. Radioactivity tests
Technology :
. Interaction tests by fluorescence, anisotropy measurement
. Search for orphan protein ligands (fluorescence)
. Interaction tests alpha-screen, HTRF, FRET, TRF, TRF, FP, etc.
. Protein-ligand interaction measurement label-free
2) Examples of functional assays
Nature of the test:
Technology:
Phenotypic screening aims to modify a phenotype without necessarily knowing the biological target responsible for that phenotype.
They are mainly performed on fixed or living cells using either :
1. A global measurement of well fluorescence, luminescence, etc... using multimode microplate readers for high throughput screening (HTS),
2. Measurements label free technologies (DMR, impedance,...),
3. Image acquisition by automated microscopy and measurement of cell parameters, cell by cell, with high information content (High Content Screening/Analysis/Screening HCS/HCA) using fluorescent probes or phase contrast imaging, etc). The HCS technology is mainly used if the studied phenotype requires a visualization of cellular substructures (compartments, organelles, nucleus, cytoskeleton...) and allows a medium or high with a 96 or 384 well plate miniaturization.
Phenotypic screens can also be carried out on single-cell living organisms (bacteria, yeast, fungi) with high throughput, or on complex living organisms (nematode, ascidium, drosophila, zebra fish,...) at throughputs adapted to each organism.
For example, phenotypic essays on living or fixed cells available in HTS or HCS format are listed below. New customized tests can be developed in collaboration with the infrastructure (see "Miniaturization of tests"):
Cell survival, proliferation, apoptosis, necrosis, mitosis
Expression of reporter genes
Quantification and/or visualization of biomarker expression
Cellular migration
Cellular injury, wound healing
Cellular traffic, autophagy
Cytoskeleton remodeling
Measurement of the effect of ligands on cells (label-free: EPIC technology, impedance...)
Measurement of second messengers (AMPc, IP1, calcium...)
Measurement of membrane potentials
Measurement of excreted proteins (chemokines, cytokines, etc.)
Viral, bacterial replication
Project request
Measurement of receptor-ligand binding on whole cells without labelling
Introduction:
The Label Free cell test, based on Corning Epic® technology, measures phenotypic changes in whole cells following a stimulus resulting in a dynamic mass redistribution of the cell (DMR).
Dynamic mass redistribution of the cell in response to a stimulus occurs in the majority of biological events, and its measurement can be performed in many applications, such as ligand binding, receptor activation or inhibition, intracellular recruitment, cytotoxicity, viral infection, endocytosis, chemotaxis..
Principle: Cells are seeded in plates coated with optical biosensors. The bottom of the plate is illuminated by wideband light, and the mass redistribution of the cells following a stimulus causes a modification of the refractive index of the cell monolayer.This change in index is detected by biosensors, and results in a variation in pm (picometers) of the wavelength of refracted light.
Protocol
To be adapted according to the test. Format: 384 wells Cells: adherent cells
1) Cells are seeded into the plate
2) Incubate 1 night at 37°C 5% CO2
3) Read baseline
4) Process cellular response data
Measurement of DMR on HEK cells overexpressing the oxytocin receptor
1) Measurement of the agonist effect of oxytocin
Format: 384 wells
Cells: HEK293 overexpressing the oxytocin receptor
2) Measurement of the antagonistic effect of L-368,899 - Format: 384 wells
Cells: HEK293 overexpressing the oxytocin receptor
In the presence of 30 nM oxytocin
Intracellular cAMP measurement
Introduction
Cyclic adenosine 3', 5'-monophosphate (cAMP) is one of the most important second messengers. It is involved in the physiological responses of neurotransmitters, hormones and drugs.
cAMP is produced from adenosine triphosphate (ATP) by membrane adenylate cyclase. The regulation of intracellular concentration of cAMP is controlled by the balance between its synthesis from ATP and its rapid degradation to 5'-AMP by phosphodiesterases (PDE).
Some GPCR receptors can control cAMP production by acting through the activation of specific G proteins, capable of stimulating (Gs) or inhibiting (Gi) its production.
Measurement of intracellular cAMP is therefore a method to quantify the effect of compounds on certain GPCRs.
Principle
The test is based on the competition between the europium-labelled cAMP and the sample cAMP for binding to antibodies against cAMP labelled with the dye ULightTM.In the absence of free cAMP, the antibody binds to the tracer cAMP, the energy emitted by the europium excited at 320 or 340nm is transferred by FRET to the molecule ULightTM which emit at 665nm, the TR-FRET signal is maximum. In the presence of free cAMP, there is competition for antibody binding, so that TR-FRET signal is decreased
Protocol
Format: 384 wells
Cells: HEK293 in suspension (possibility to work on other cells)
Receiver: Gαs coupled GPCR
1. Distribution of cells in the plate
2. Stimulation with compounds of interest
3. Addition of tracer cAMP and anti-cAMP antibody solutions ULightTM
4. Measurement
Excitation wavelength: 320 nm
Emission wavelength 1: 615 nm
Emission wavelength 2 : 665 nm
Vasopressin receptor activation and measurement of the associated cAMP signal
Format: 384 wells
Cells: HEK293 overexpressing the vasopressin receptor AVPR2
Intracellular IP1 determination
Introduction
The detection of intracellular second messengers such as IP1, produced following the activation of Gq proteins coupled receptors, can be achieved by HTRF® technology.
HTRF® technology (http://www.cisbio.com/) is based on the basic principle of FRET. The properties of the fluorophores used provide many advantages.
Principle
Detection of IP1 relies on a competition assay. The anti-IP1 antibody (coupled to a donor fluorophore) provided by the kit recognizes the Fluorescence acceptor-tagged IP1 and competes with intracellular IP1 in a dose-dependent manner.
LiCl is added to the reaction buffer to allow accumulation of the IP1 produced in the cells.
Protocol :
Format: 384 wells
Cells: HEK293 in suspension (possibility to work on other lines)
1) Distribution of cells in the plate
2) Stimulation
3) Incubation at 37°C (time varies according to cell type and receptor)
4) Addition of HTRF reagents
5) Incubation 1h at room temperature
6) Reading
Excitation wavelength: 337 nm
Emission wavelength 1: 665 nm (emission of donor fluorophore)
Emission wavelength 2: 615 nm (emission of the accepting fluorophore)
Determination of intracellular IP1 following stimulation of the oxytocin receptor
HEK293 cells overexpressing the oxytocin receptor Analysis: S = 665 nm signal / 615 nm signal
1) Standard curve
1) Measurement of agonistic effect of oxytocin and carbetocin
3) Measurement of the antagonistic effect of the compound L-869,899 in the presence of 30 nM agonist
Intracellular calcium determination
Introduction
Calcium is involved in many physiological processes (release of neurotransmitters, muscle contraction, blood coagulation...) and in cell signalling where it acts as a second messenger following the activation of heterotrimeric Gq proteins via receptors coupled to G proteins.
This test dynamically monitors changes in cytoplasmic calcium concentration in living cells.
Principle
Calcium measurements are performed using a calcium-sensitive fluorescent probe (Indo1 or Fluo4). Depending on the nature of the probe, its binding to calcium induces a variation in its fluorescence intensity and possibly a shift in its emission spectrum:
The probes are made permeable by the addition of an acetoxymethylester (AM) group, and can thus pass the plasma membrane of the cells. The AM part is released inside the cell by intracellular esterases.
Measurements are performed on a semi-robotized reader (Flexstation3® Molecular Devices) that allows real-time detection of intracellular calcium release.
Protocol
Calcium probe:
Indo1-AM (λex338nm / (λem1401nm, (λem2475nm)
Fluo4-AM ((λex494nm / (λem516nm)
Format: 96 or 384 wells
Cells: HEK293 in suspension (possibility to work on other lines)
1. Load cells with the probe
2. Suspend cells and distribution in plates
3. Basal signal measurement
4. Treatment of cells with basal compounds and recording
Up to 3 consecutive treatments can be carried out.
Stimulation of the oxytocin receptor and measurement of the associated calcium signal
Format: 384 wells
Cells: HEK293 overexpressing oxytocin receptor
Determination of cytokine secretion by human mononuclear cells
Introduction
Cytokines, secreted by the effector cells of the immune system, have a major role in most chronic inflammatory diseases.
Their quantification is therefore a relevant biological target in the search for anti-inflammatory active ingredients.
Principle
Determination of cytokines secreted by human mononuclear cells can be performed by ELISA immuno enzymatic assay or with HTRF technology.
1. ELISA
The proteins to be determined, bound to a capture antibody, are detected by a biotin-streptavidin complex coupled to peroxidase, which in the presence of its substrate allows the development of a colorimetric reaction detected by absorbance at 450 nm. The measured absorbance is proportional to the concentration of the cytokine measured.
2. HTRF (CISBIO INTERNATIONAL)
Two fluorophores donor (europium cryptate) / acceptor (XL665) partners are fused respectively to an antibody specific to the protein to be measured. The delayed time energy transfer (TR-FRET) between the fluorophore pair is proportional to the concentration of cytokine present in the sample.
www.cisbio.com . This test is homogeneous and requires only small amounts of proteinProtocol
Mononuclear cells isolated from human peripheral blood
1. Processing
Formats: 24 or 96 wells
Stimulation: 5 µg/mL LPS or 5 µg/mL PHA
Reference molecules: Dexamethasone; Rolipram
Incubation: 24H at 37°C under 5% CO2.
2. Cytokine detection
ELISA assay: 96-well format / absorbance measurement at 450 nm
HTRF assay: Format 384 wells
Excitation wavelength: 337 nm
Emission wavelength 1: 615 nm (emission of donor fluorophore)
Emission wavelength 2: 665 nm (emission of the accepting fluorophore)
Secretion of TNF-α by human mononuclear cells in the presence of anti-inflammatory compounds
The assays for TNF-α, IL1-ß, IL-6 and IL-8 have been already developed by ELISA. The assays for TNF-α, IL1-ß, IL-6, IL-8, IL-10 and INF-γ were developed by HTRF.
Other cytokines can be tested on request.
Project request
Introduction
The Label Free biochemical test, based on Corning Epic® technology, makes it possible to measure, without any labelling, the binding of a ligand to a target protein.
Principle
The target protein is immobilized in plates coated with optical biosensors.
If required, it is possible to include a spacer group to provide better accessibility to the protein.
The bottom of the plate is illuminated by wide spectrum light, and mass changes related to the attachment of a ligand to the target protein cause a change in the refractive index of the refracted light. This change in index is detected by biosensors, and results in a variation in pm (picometers) of the wavelength of the refracted light. A reference area in the well prevents immobilization of the target, and thus allows results to be generated that only represent the effects of ligand binding on the protein.
Protocol
To be adapted according to the test
Format: 384 wells
Pre-activated or operator-activated plates
1) Immobilization of the target on the plate, 1h at room temperature
2) Incubation 1 night at 4°C
3) Washing with PBS
4) Incubation 4h at room temperature
5) Reading the baseline
6) Processing
7) Plate reading
Measurement of the binding of biotin to streptavidin by label free technology
Pre-activated plates - Streptavidin 75 µg/mL
Biotin: 25 nM to 40 µM
1) Measurement of streptavidin immobilization (baseline)
2) Detection of the biotin-streptavidin binding
We offer support and preliminary feasibility study to miniaturize assays in order to make them compatible with high throughput screening, while reducing costs and increasing their statistical robustness.
Assays are miniaturized to microplate format (generally 96 or 384 wells) and the experimental procedure is adapted to allow the processing of samples by pipetting robots and to screen a large number of compounds (alone or in combination). Biological effects are quantified by various measurements of fluorescence, luminescence, radioactivity or imaging.
Monitoring bacterial strain growth
Introduction / Principle
This assay identifies potential inhibitors of bacterial growth.
Growth of a bacterial strain is monitored over several hours by turbidimetry in the presence of the molecules to be tested.
Bacterial concentration is proportional to the absorbance measured at 620 nm.
Protocol
Format: 96 wells
Bacterial strain: To be defined
Concentration of the molecules to be tested: To be defined
Incubation: To be defined
Optical density measurement at 620 nm as a function of time
Introduction
Studying cytotoxicity of a compound with modulating activity on cell response is essential.
It allows to control that the active molecule does not induce any change in cell viability compared to untreated cells.
Several cell lines are available for cytotoxicity study
Principle
Several cell viability tests are proposed
1. MTT Test
The test is based on the cleavage by mitochondrial succinate dehydrogenase of a yellow tetrazolium salt into blue formazan crystals.
The reaction is quantified by measuring the absorbance at 560 nm of the crystals solubilized in an organic solvent that is proportional to the number of living cells.
2. Test WST-1 www.ozyme.fr
The test is based on the cleavage by mitochondrial succinate dehydrogenase of a red tetrazolium salt into yellow formazan crystals.
The reaction is quantified by measuring the absorbance at 450 nm of the soluble crystals in the culture medium, which is proportional to the number of living cells.
Unlike MTT, WST-1 is a tetrazolium salt capable of being cleaved by living cells into soluble formazan, avoiding subsequent solubilization in an organic solvent.
3. Glo Luminescent Cell Titer assay (PROMEGA) www.promega.com
Assay is based on the mono oxygenation of luciferin to oxyluciferin by luciferase in the presence of Mg 2+ and ATP. The reaction is quantified by measuring the luminescent signal which is proportional to the ATP present in the medium and therefore to the metabolic activity of the cells.
4. IncuCyte (ESSEN BIOSCIENCE) www.essenbioscience.com
This device makes it possible to carry out kinetics of cell proliferation under conventional culture conditions under controlled atmosphere by quantifying cells confluency.
Protocol
Format: 96 or 384 wells
Seeding of cells 24H before treatment
Reference molecule : Chlorpromazine
Incubation 48H at 37°C (possibility to adapt the incubation time to the biological model)
Carrying out cell viability assay
Evaluation of Chlorpromazine cytotoxicity on HepG2 (ATCC HB-8065)
IncuCyte (ESSEN BIOSCIENCE)
Protein kinases are involved in the regulation of numerous cellular processes, often in response to an external stimulus. This family of enzymes has become one of the most important suppliers of drug targets. The number of approved small-molecule protein kinase inhibitors (PKIs) continues to grow and more then 50+ drugs have reached the US market, 85% of which are used for the treatment of malignancies. More than 200 orally effective PKIs are currently in clinical trials worldwide (a complete and updated listing of PKIs in clinical trials can be found at www.icoa.fr/pkidb/).
Natural or synthetic chemical compounds and also semi-purified fractions of natural extracts can be analyzed on to discover new PKIs. A structure-activity study can be performed to identify a lead compound. The binding mode of the hit compound to its kinase target can also be evaluated by an ATP competition assay.
Kinase assay:
Kinase enzymatic activities were assayed in 384-well plates using the ADP-GloTM assay kit (Promega, Madison, WI). This assay is a luminescent ADP detection assay that provides an homogeneous and high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.
Reactions were carried out in a final volume of 6 µl for 30 min at 30°C in appropriate kinase buffer, with either protein or peptide as substrate in the presence of 10µM ATP. After that, 6 µl of ADP-GloTM Kinase Reagent was added to stop the kinase reaction. After an incubation time of 50 min at room temperature (RT), 12 µl of Kinase Detection Reagent was added for one hour at RT. The transmitted signal was measured using the Envision (PerkinElmer, Waltham, MA) microplate luminometer and expressed in Relative Light Unit (RLU). Kinase activities are expressed in % of maximal activity, i.e. measured in the absence of inhibitor.
*(A) Primary kinase-based screening and * (B) determination of IC50
(A) Compounds are first tested at two concentrations (1 µM and 10 µM) against a panel of disease-related kinases. (B) Subsequently, compounds displaying more than 50% inhibition at 1 or 10 µM were next tested in duplicate over a wide range of concentrations (usually from 0.0003 to 10 µM) against the selected kinases and IC50 values were determined from the dose response curves using GraphPad Prism.
Primary Screening of chemical library
Determination of IC50
* ATP competition test by ADP-Glo method
To study the mechanism of inhibition of the hit compounds, we performs kinetic experiments by varying both ATP levels and inhibitor concentrations to draw a double-reciprocal plot of the Michaelis-Menten equation (also known as the Lineweaver-Burk).
Kinase panel
Customers will select the kinases from the following list: 1. CDK2/CyclinA (CMGC), 2. CDK5/p25 (CMGC), 3. CDK9/CyclinT (CMGC), 4. CDK10/CyclinM (CMGC), 5. GSK3α (CMGC), 6. GSK3ß (CMGC), 7. CLK1 (CMGC), 8. DYRK1A (CMGC), 9. PIM1 (CAMK), 10. CK1ε (CK1), 11. NEK6 (Other), 12. NEK7 (Other), 13. ABL1 (TK), 14. EGFR (TK), 15. VEGFR-2 (TK), 16. JAK3 (TK), 17. Haspin (Other), 18. RIPK3 (TKL), 19. PfGSK3 (from Plasmodium falciparum), 20. LmCK1 (from Leishmania major).
The panel of kinase available reported on the representation of
the human kinome.
The codes reported on this figure indicate the subclasses of protein kinases: CMGC for CDKs, MAP kinases, GSK, and CDK-like kinases; AGC for protein Kinase A, C, and G families (PKA, PKC, PKG); CAMK for Ca2+/calmodulin-dependent protein kinases; CK1, Cell/Casein Kinase 1; STE, STE Kinases (Homologs of yeast STErile kinases); TKL, Tyrosine Kinases-Like; TK, Tyrosine Kinases.
To validate each kinase assay, model inhibitors were used under the same conditions than the tested compounds. For example: Indirubin-3'-oxime for CDK2/CyclinA, CDK5/p25, CDK9/CyclinT, RnDYRK1A and MmCLK1; SGI-1776 for Pim1; Staurosporine from Streptomyces sp. for CK1ε; Tofacitinib for JAK3; Imatinib mesylate for ABL1; CHR-6494 for HASPIN; and GSK'872 for RIPK3.
Project request