In Vitro Acid Phosphatase Assay for Cell Viability | PraxiLabs

In Vitro Acid Phosphatase Assay for Cell Viability Virtual Lab Simulation

Biology | Toxicology | Biochemistry | Proteomics | Pharmacology



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General Aim

This experiment aims at quantifying the amount of acid phosphatase activity on the cell membrane of viable cells using microplate reader.

Method

In Vitro Acid Phosphatase assay using microplate reader

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 Acid Phosphatase assay solution. Stopping the reaction and developing the color will be calorimetrically measured.

  • Analyze color intensity of the reaction by microplate reader and analyze resulted data.

  • Represent and graphically locate the IC50 of the tested nanoparticles.

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

Traditionally, the in vitro determination of toxic effects of unknown compounds or nanoparticles has been performed by counting viable cells after staining with a vital dye. Alternative methods used are measurement of radioisotope incorporation as a measure of DNA synthesis, counting by automated counters and others which rely on dyes and cellular activity. The acid phosphatase assay is a means of measuring the mass of cells via cell-membrane associated acid phosphatase. The acid phosphatase method is simple, accurate and yields reproducible results. The key component is p-Nitrophenyl phosphate. Solutions of p-Nitrophenyl phosphate in medium or PBS without phenol red are colorless.

 

Cell-membrane associated acid phosphatase cleaves the p-Nitrophenyl phosphate substrate yielding a yellow colored compound (p-Nitrophenol) which is soluble in aqueous solutions. The resulting yellow solution is measured spectrophotometrically. An increase or decrease in cell numbers results in a simultaneous change in the amount of substrate converted, indicating the degree of cytotoxicity caused by the tested nanomaterial. 

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