Cytotoxicity

Cytotoxicity is the quality of being toxic to cells. Cells exposed to a cytotoxic compound can respond in a number of ways. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis; they can stop growing and dividing; or they can activate a genetic program of controlled cell death, termed apoptosis.

There are many ways to measure cytotoxicity, but most involve assessment of cell membrane integrity. Membrane integrity can be evaluated by using vital dyes (such as trypan blue or propidium iodide), by protease biomarkers, with MTT or MTS redox potential assays, or by measuring ATP content. Many of these assays involve colorimetric, fluorescence, or luminescence detection.

With one of the widest ranges of detection technologies on the market today, Molecular Devices can provide the bioanalytical and analytical products that you need to conduct cytotoxicity measurements, from cytometry and high-content screening systems to multi-mode microplate analysis systems to high-throughput cellular screening systems.

Cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death.

Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beetle luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.

Atomic Absorption Spectroscopy (AAS)

Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.

In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples used in pharmacology, biophysics and toxicology research.

Atomic absorption spectroscopy was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.

The modern form of AAS was largely developed during the 1950s by a team of Australian chemists. They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Chemical Physics, inMelbourne, Australia.

Atomic absorption spectrometry has many uses in different areas of chemistry such as:

• Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen
• Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product
• Water analysis: Analyzing water for its metal content.

Atomic Absorption Theory

Atomic absorption spectroscopy relies on the Beer-Lambert law to determine the concentration of a particular analyte in a sample. The absorption spectrum and molar absorbance of the desired sample element are known, a known amount of energy is passed through the atomized sample, and by then measuring the quantity of light it is possible to determine the concentration of the element being measured.

Instrumentation

In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used nowadays are flames and electrothermal (graphite tube) atomizers. The atoms should then be irradiated by optical radiation, and the radiation source could be an element-specific line radiation source or a continuum radiation source. The radiation then passes through a monochromatorin order to separate the element-specific radiation from any other radiation emitted by the radiation source, which is finally measured by a detector.

Atomizers

The atomizers most commonly used nowadays are (spectroscopic) flames and electrothermal (graphite tube) atomizers. Other atomizers, such as glow-discharge atomization, hydride atomization, or cold-vapor atomization might be used for special purposes. Here are some atomizers:

• Flame Atomizers
• Electrothermal Atomizers

Specialized atomization techniques

While flame and electrothermal vaporizers are the most common atomization techniques, several other atomization methods are utilized for specialized use.

• Glow-discharge atomization
• Hydride atomization
• Cold vapor atomization

Radiation sources

We have to distinguish between line source AAS (LS AAS) and continuum source AAS (CS AAS). In classical LS AAS, as it has been proposed by Alan Walsh, the high spectral resolution required for AAS measurements is provided by the radiation source itself that emits the spectrum of the analyte in the form of lines that are narrower than the absorption lines.

• Hollow cathode lamps
• Electrodeless discharged lamps
• Deuterium lamps
• Continuum Sources

Spectrometer

As already pointed out above, there is a difference between medium-resolution spectrometers that are used for LS AAS and high-resolution spectrometers that are designed for CS AAS. The spectrometer includes the spectral sorting device (monochromator) and the detector.

• Spectrometers for LS AAS
• Spectrometers for CS AAS