Annexin V Assay Virtual Lab Simulation | PraxiLabs

Annexin V Assay Virtual Lab Simulation

Biology | Toxicology | Biochemistry | Pharmacology

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Annexin V Assay Virtual Lab

General Aim of Annexin V Assay Experiment

Annexin V assay experiment aims at detecting and quantifying apoptotic and necrotic cells through Annexin-V binding and Propidium Iodide (PI) uptake using fluorescent microscope.

Annexin V Assay Experiment Method

In Vitro Annexin V Binding/Propidium Iodide Uptake Assay using Fluorescence Microscope.

Learning Objectives of Annexin V Assay Virtual Experiment

  • Successfully handle the required instruments and consumables needed in the experiment.

  • Check the confluence and count cells under the microscope.

  • Dilute the cells to a specific count suitable for seeding in the 96-well plate.

  • Calculate the concentration of tested chemicals and prepare the calculated doses in the cell culture medium.

  • Treat cells with the cytotoxic agent(s) or nanoparticles and observe under the microscope.

  • Harvest cells with Annexin binding buffer ( A Annexin-V / Propidium Iodide buffer).

  • Analyze cells by fluorescent microscope and analyze resulted data. Represent and interpret the resulted data graphically using dot plots.

Theory of Annexin V Assay Experiment

Cytotoxicity is the quality of being toxic to cells. Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers, as in Nanotechnology, can either look for cytotoxic nano-based materials, if they are interested in developing a nanomedicine that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput nanoparticle screens for unwanted cytotoxic effects before investing in their development as a nanomedicine. 

Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. 

Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity. 

Cytotoxicity can also be monitored by measuring the reducing potential of the cells using a colorimetric reaction, or using ATP content as a marker of viability. Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction. A label-free approach to follow the cytotoxic response of adherent animal cells in real-time provides the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays.

Annexin V Assay Principle

Annexin V/PI staining principle depends on the following: The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. 

However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane. Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. 

Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane. However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells.

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