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Scratch Wound Healing assay

Introduction / Principle

Cellular monolayer healing is monitored by an automated microscopy machine: the IncuCyte (ESSEN BIOSCIENCE) (quantification of cellular confluence).


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



Monitoring of healing on HaCaT cells  

Project request
Molecular screening on purified targets ("Target-based")

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:

  • Various enzymes (proteases, kinases, phosphatases, methyl transferases, viral enzymes, etc.)
  • Protein-protein interactions/functional aggregation



  •   Absorbance (colorimetry), fluorescence, luminescence or in radioactivity

Project request
Phenotypic screening on cells or living organisms ("Phenotypic-based")

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
Measuring the effect of ligands on cells (label-free)

Measurement of receptor-ligand binding on whole cells without labelling


 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.


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

Project request
Measurement of second messengers (cAMP, IP1, calcium...)

Intracellular cAMP measurement



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.




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


 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 


 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 ( is based on the basic principle of FRET.  The properties of the fluorophores used provide many advantages.


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


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.



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.


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

Project request
Measurement of excreted proteins (chemokines, cytokines, etc.)

Determination of cytokine secretion by human mononuclear cells



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.



Determination of cytokines secreted by human mononuclear cells can be performed by ELISA immuno enzymatic assay or with HTRF technology.


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.