Live cell imaging and analysis

Our research Unit acquired instruments for live cell functions analysis:

Incucyte® SX5 Live cell Imaging System allows to acquire and view images over time (Fluorescence and HD phase imaging modes, Minimize cell disturbance with a mobile optical train).

XCelligence Agilent uses live, simultaneous, and real time biosensor impedance-based and image-based measurements.

In addition, we acquired the Lonza’s 4D-Nucleofector System, a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates including plasmid DNA and siRNA.

Coagulation analysis and platelet function

Our research Unit has invested in technologies allowing coagulation analysis and characterization of human and mouse platelet functions.

Coagulation analysis

ROTEM®delta

The ROTEM delta is an integrated all-in-one system allowing the measurement of thromboelastometry parameters in platelet poor plasma or whole blood. This viscoelastometric method measures the interactions of coagulation factors, inhibitors and cellular components during the phases of clotting and subsequent lysis over time during a one-hour time reaction at 37°C. This system can assess a series of coagulation parameters such as clotting time (CT), clot formation time and firmness (CFT and MCF) and also fibrinolytic parameters as maximal lysis (ML) and lysis time and rate (LT, CLR).

StarMax

The coagulation analyzer StartMax is a semi-automated benchtop analyzer for all clotting assays from routine to specialized assays. Measurements can be qualitative or quantitative using small volumes of human or mouse materials.

Platelet function

The “platelet platform” has many applications including:

• Basic research studies on the physiologic role of platelets in hemostasis and their pathologic role in thrombosis
• Identification of platelet defects in patients
• Clinical studies: diagnostic markers and platelet responsiveness to therapeutic agents
• Bioengineering: analysis of the biocompatibility, hemostatic toxicity of polymers and devices; development of new tracers.

The platform can acquire the following parameters:

• Hematological parameters can be analyzed by a human or mouse blood analyzer. Micro-sampling from whole blood allows real cell volume measurement:

– Sysmex XE for platelet quantification and volume

– ABX Pentra60 Horiba for Human hemocytometry

– SCIL Vet ABC for mouse hemocytometry

• In vitro bleeding time is acquired with a PFA100 Siemens
  Platelet phenotype is analyzed by flow cytometry (see above), immunoblot and immunoprecipitation.
• Platelet activation is assessed by analyzing expression of activation markers by flow cytometry
  Platelet aggregation is measured by light aggregometry (8 channels aggregometer: APACT 40004, Elitech)

• Platelet procoagulant activity is assessed by thrombinography on platelet rich plasma. It consists in measuring the catalytic activity of platelets on the formation of thrombin.

• Platelet adhesion in static conditions is performed by colorimetric analysis.
• Shear-induced platelet activation is analyzed with “Couette” devices (Hospital hematology laboratory). The principle is to subject platelets to increasing shear rates in order to reproduce the conditions of a stenosed vessel. Platelet activation is measured as the disappearance of individual platelets.

In vitro cell adhesion, thrombosis and thrombolysis can be studied under flow Microfluidic flow chambers. The laboratory has the possibility to analyze the capacity of platelets to form thrombi in whole blood under flow condition with platelets being labeled with a fluorescent probe (DiOC6 or rhodamine). Two types of flow chambers are available: the classical Maastricht chamber and the small volume dispensable chambers from Cellix (Dublin, Ireland) either coated with purified proteins or covered by an endothelial cell monolayer. Modulation of flow conditions is possible with the use of Exigo and/or Myrus pumps and offer the possibility to work at defined shear stress.

A specific protocol developed by LVTS T6 allows real time analysis of clot formation and lysis in human whole blood under arterial or venous flow rates and offers a way to evaluate the lytic efficacy of drugs during thrombolysis.