fr | en
menu

Assay miniaturization

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.

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

 

Technology:

  •  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
Evaluation of the cytotoxic effects of a compound

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)


Project request
Reporter genes expression

<To be filled out>

Project request
Quantification and/or visualization of biomarker expression

« To be filled out »

Project request
Cell migration

« To be filled out »

Project request
Blessure cellulaire, cicatrisation

Project request
Cell traffiking, autophagy

« To be filled out »

Project request
Cytoskeleton remodelling

« To be filled out »

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

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).

 


www.corning.com


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.


www.corning.com



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


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

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 ULight™.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 ULight™ 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 ULight™

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.