In Vitro Fluorescein Diacetate/Propidium Iodide (FDA/PI) Staining Assay

In Vitro Fluorescein Diacetate/Propidium Iodide (FDA/PI) Staining Assay Virtual Lab Simulation

Biology | Toxicology | Biochemistry | Proteomics | Pharmacology



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General Aim

This experiment aims at calculating the viability percent of cultured adherent cells after excitation of free fluorescein cleaved by esterase enzymes in live cells using fluorescent microscope.

Method

In Vitro Fluorescein Diacetate/Propidium Iodide (FDA/PI) Staining Assay using fluorescence microscope

Learning Objectives (ILO’s)

  • 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.

  • Treatment of cells with the FDA/PI working solution in cell culture medium.

  • Count cells with bright green fluorescence (for viable cells), as well cells of bright red fluorescence (for dead cells) using fluorescence microscope.

  • Calculate the viability percent of viable and dead cells.

  • Represent the resulting data graphically and present it.

Theoretical Background/Context

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.

Principle of Work

Live/dead staining can be performed with FDA and PI. FDA is taken up by cells that convert the non-fluorescent FDA into the green fluorescent metabolite fluorescein. The measured signal serves as an indicator for viable cells, as the conversion is esterase dependent. In contrast, the nuclei staining dye PI cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membranes and intercalates with the DNA double helix of the cell.

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