CELLULAR ASSAYS

Porsolt maintains a panel of over 100 validated cell-based assays allowing the quantification of key phenotypic and molecular events at the single-cell level. Most of the cellular assays listed below can be transferred to a different biological model, or adapted to a different detection platform, according to your need. Learn about our assay development services.

MOLECULAR READOUTS

PHENOTYPIC READOUTS: MORPHOLOGICAL CHANGES

Angiogenesis

Porsolt offers to identify and characterize compounds modulating angiogenesis through a quantitative in vitro endothelial tube formation assay. In addition, a range of molecular assays is available to explore the mechanism of action of compounds either activating or inhibiting angiogenesis.

Our assay is based on the ability of endothelial cells to form capillary-like tubes in a co-culture model of fibroblasts and GFP-expressing HUVECs:

> Cells cultured in a specifically designed medium
> Angiogenesis is monitored by live content time-lapse imaging for the identification and visualization of HUVEC tube formation
> Tube formation is analyzed using an appropriate angiogenesis module, for robust determination of the total tube length
> Our angiogenesis assay enables to characterize compounds' effects in both dose- and time-dependent manner.

To better understand the mechanim(s) of action of angiogenic modulators, we offer to monitor the status of specific signaling pathways related to angiogenesis, such as H1F1α, Smad, P38, PI3K...

In addition to direct measurements of neuritogenesis, protein markers such as actin, tubulin, GSK3B, ERK, Jun, c-Fos, etc..., can be quantified at the single-cell level by high-content imaging to study specific signaling pathways/mechanisms of action.

>>> Learn more about our services in neurobiology

 

VEGF-induced angiogenesis HUVEC/fibroblasts co-culture model.

Fluorescence images of GFP-expressing HUVECs are segmented with an automated angiogenesis analysis module, and white segments correspond to the vascular tube-like structures are quantified and total tube length is thus determined.

HUVEC tube formation is kinetically monitored by live content imaging over a 9 days post-treatment period. VEGF dose-dependently induces HUVEC tube formation. The VEGF receptor tyrosine kinases 1, 2 and 3 inhibitor, Axitinib, exerts an anti-angiogenic activity thereby suppressing VEGF-induced pro-angiogenic effects.

Neurite outgrowth

Neuritic growth is a key readout to monitor neuronal function and health. Porsolt has developed a range of in vitro neurite outgrowth assays, to be performed in a variety of cell lines or primary cell cultures, in order to identify and characterize compounds inducing neuroprotection, neuroregeneration, neurotoxicity, or modulating neuritogenesis.

KINETIC NEURITE OUTGROWTH ASSAY

> Kinetic & label-free neurite outgrowth monitoring performed by live-content imaging

> Segmentation of phase-contrast images & quantification of total neuritic network

> Neuroprotective or neurotoxic effects characterized over several days

ENDPOINT NEURITE OUTGROWTH ASSAY

> Automated high-content imaging, using confocal microscopy

> Single-cell image analysis: neuritic growth can be ascribed to single cells using segmentation mask parameters

> Multiplexed detection with neuronal markers enables to measure neurite outgrowth in specific subpopulations

Segmentation mask of differentiated PC12 cells: neurites are represented as thin lines extending from cell bodies.

Cell migration| Cell invasion & adhesion

Cell migration is central to key biological functions such as embryogenesis, immune response and inflammation. But it also participates in many disorders such as vascular diseases and cell cancer metastasis. We developed standardized, sensitive and robust cell migration assays to study its underlying mechanisms and test novel therapeutic strategies.

 MIGRATION OF ADHERENT CELLS: THE WOUND HEALING ASSAY

> No reagents: cell migration is triggered by removing a strip of cells at the center of the well, using a 96-well wound maker tool.

> Kinetic monitoring: cell migration can be monitored over several days by label-free live-content imaging.

> Quantitative: cell migration is quantified by measuring cell density within the wound.

> Cell invasion can be addressed using similar approach based on matrigel matrix.



MIGRATION OF NON-ADHERENT CELLS: THE TRANSWELL MIGRATION ASSAY

> 96-well Boyden chamber assay: a gold-standard for in vitro chemotaxis measurements

> High-throughput: total number of cells and the number of migrating cells are quantified by flow cytometry.

Case study : Transwell migration of monocytic cells THP-1 induced by Fractalkine (FKN) and reversed by Wortmannin (Wort)=>

Cell growth | Cell proliferation

Assessing in vitro cell proliferation is a key mean for monitoring cellular health but also for the functional screening of drug candidates. Porsolt offers different options to identify and characterize compounds either activating or inhibiting cell growth and proliferation.

PROLIFERATION INDEX ASSAY

Porsolt proliferation index assay is both simple and robust, being based on the dilution rate of a fluorescence membrane dye:

> The dye is compatible with multiplexed analysis

 Fast turnaround: assay is performed by high-throughput flow cytometry

Accurate: dilution rate of fluorescent probe is a direct readout of the number of cell divisions. Data is not affected by variations in cell number per well or cell metabolic activity.

KINETIC CELL PROLIFERATION ASSAY

We use live-content imaging to monitor cell proliferation over time:

> Kinetic & label-free proliferation assay (no reagent needed)

No hidden drug effects: cells are monitored over several days

Accurate compound profiling: time-dependent EC50 values

Multiplexing with cytolysis detection

>>> Cell proliferation can also be assessed by flow cytometry or high-content imaging, based on BrdU/EdU incorporation to label DNA-replicating cells

HIGH-CONTENT ANALYSIS OF CELL GROWTH AND PROLIFERATION MARKERS

We offer to quantify protein markers involved in cell growth and proliferation such as Ki67, Histone H3, p21, PCNA... at the single-cell level in 2D & 3D cellular models, using high-content imaging methodologies.

>>> Learn about our drug discovery services in oncology and approach for predictive toxicology.