CELL DEATH, APOPTOSIS, AUTOPHAGY

Because cell-death related assays are of primary importance in the context of drug discovery and cytotoxicity testing, Fluofarma developed a range of assays designed for high-throughput multiplex screening. Compatible with different high-content analysis platforms, our assays can be adapted to the cellular model of your choice.

CYTOLYSIS ASSAYS

 

Because of their relative low cost and compatibility with high-throughput screening, cytolysis assays represent a cost-efficient approach to identify cytotoxic compounds. In addition, when multiplexed, our cytolysis tests enable to characterize cell death signaling induced by drug candidates.

 

> High-throughput cytolysis assay: performed by flow cytometry, suited for large screening campaigns and multiplexed analysis.

> Kinetic cytolysis assay: dynamic measurements by  live-content imaging, commonly used to differentiate cytostatic from cytotoxic drug candidates.

Learn about our drug profiling & MoA services.

APOPTOSIS ASSAYS

Apoptosis is a highly regulated process of programmed cell death involving the activation of specific signaling cascades leading to cell shrinkage, nuclear condensation and fragmentation. Fluofarma offers to detect apoptosis through several methods:

 

> Proprietary biosensors for the reagentless quantification of caspase-3 activity and calpain-activity by  high-throughput flow cytometry

> Caspase 3/7 activation can be measured in any cell type, either by high-throughput flow cytometry or high-content imaging

> In addition to classical measurements of apoptosis, we offer to quantify protein markers involved in apoptosis, such as γH2AX, p53, P-AKT... by high-content imaging for single-cell level analysis.

AUTOPHAGY-RELATED ASSAYS

 

In mammals, autophagy involves a lysosomal degradation pathway, which is essential for differentiation, survival, development and homeostasis.

Autophagy is characterized by induction and translocation of LC3B, autophagosome formation, lysosomal fusion and degradation of autophagosome content.

The whole process is tightly regulated by the PI3K-Akt-mTOR, LKB1-AMPK-mTOR and p53 pathways.

Fluofarma offers different assays to study the different aspects of autophagy by high-content imaging:

> LC3B assay: detection of endogenous LC3B specifically incorporated into autophagosomes.

> Lysosome-related markers such as lysotraker are used to evaluate lysosomal activity

> Other markers involved in the regulation of autophagy can be studied at the single-cell level with subcellular resolution, such as mTOR, PARP cleavage, protein ubiquitination...

MITOCHONDRIAL MEMBRANE POTENTIAL ASSAYS

MCF7 cells. Nuclei are stained with DAPI (blue) and mitochondrial membrane potential is detected with TMRM (red)

MCF7 cells. Nuclei are stained with DAPI (blue) and mitochondrial membrane potential is detected with TMRM (red)

 

Collapse of mitochondrial membrane potential is a key readout of cell death, as it can be observed either early (induced by ionic or oxidative stresses) or later during apoptosis (upon caspase 3 activation).

Our mitochondrial membrane potential assays, based on the use of lipophilic probes, are validated on different platforms compatible with multiplexed analysis:

> Flow cytometry: a high-throughput approach compatible with non-adherent cells

> FLIPR Tetra®: to monitor mitochondrial potential at ultra-high throughput in homogeneous cell populations

> High-content imaging: to combine mitochondrial membrane potential measurements with morphological parameters or multiplexed biomarkers such as caspase 3, c-Jun, p38...

           Learn about our preclinical services in oncology, neurobiology and predictive toxicology