What is a cell proliferation assay?

ASSAY | Tebu Bio

A cell is the basic functional and structural element that makes up the tissues and organs of living beings. Often considered as the smallest part of a living organism, it is made up of many other units, each with their own function.

Human cells vary in size, but they are always small. Even the largest of them, the fertilised egg, is too small to be seen with the naked eye.

Cell division

Living beings are created at the base by a single cell which divides by an iterative phenomenon (called mitosis) to compose the human body. Each cell operates independently but in line with all the others. Cells communicate with each other via proteins called cyclins. There are different types of cyclins that tell the cell whether it should divide or not.

Cells divide and multiply for several reasons. For example, to repair wounds and replace damaged or dead cells. They also multiply to make living organisms grow.

We grow because our cells become more and more numerous, not because they become giant. There are about two trillion cell divisions a day in the human body.

Cell division takes place in different phases grouped under the name of cell cycle. The cell cycle has 2 main stages: interphase and mitosis (or meiosis).

Cell culture

Cell culture is a laboratory technique that makes it possible to bring cells of various natures and origins to life in vitro, to modify their properties or to increase their number, in order to have them in large quantities for what we want to do. Cell culture is not an end in itself, but an indispensable tool in cell biology.

It is a question of obtaining a type of living cell, of finding a means of making it survive in the artificial conditions of the laboratory, at best of making it proliferate, while maintaining acceptable stress conditions.

When culture cells are obtained, we can then experiment on them, it is possible to measure their reaction to several manipulations, to observe their comportment in different situations or confronted to many substances and to observe it and to determine if the result will be the same if they were put in front of such situation inside a living body.

But before determining it, we need a high number of cells and for this purpose, many types of proliferation assay exist to determine cell viability, activity and modification of their population.

Cell proliferation assay

A cell proliferation assay is an in vitro test used to measure the number of dividing cells. The watch of cell division is essential for several applications including the optimization of cell culture and the determination of the level of activity of cytokines (molecules secreted by many cells such as white cells and phagocytes which play a role in cellular immune response) and growth factors (proteins that encourage cell development and proliferation).

It can also be used to evaluate the enzyme activity (quantity of enzyme capable of catalysing the transformation of a substrate into a given produce) , the integrity of the cell membrane (if the membrane(s) of the cell has been modified or broken) and the production of ATP (molecule that provides energy to the cell) and other factors.

The results of these tests can be seen through the evolution of luminescence, fluorescence or colorimetry of the sample. Cytotoxicity (ability of a specialised cell to destroy other cells) tests are often used to determine the effects of a drug or of a treatment and they are precious tools for the research of new therapeutic agents as well as to help develop our knowledge about healthy cell’s mechanics.

Cell proliferation assays are mainly divided into four methods: metabolic activity assays, cell proliferation marker assays, ATP concentration assays, and DNA synthesis assays. The method you choose should depend on the type of cell you are studying, your research protocols, and the type of information you are looking to gain from the cell proliferation assay.

Metabolic activity assays

Detecting the metabolic activity of a cell population can reflect the condition of cell proliferation. Cell viability tests such as MTT is a method using colorimetry. It is used to rapidly determine the number of living cells and then the ratio between living and death cells. It is useful to determine the toxicity of a product, for example, if there is a higher number of death cells after the introduction of a substance, it can be considered as toxic.

The principle: a substance (here tetrazolium salt) is introduced, when this product encounters mitochondria respiratory enzyme, it is transformed into formazan, and turns purple. In this case, the purpler the sample the more living cells it contains.

Cell proliferation marker assays

Some antigens only exist in proliferating cells, while non-proliferating cells lack these antigens. Cell proliferation marker assays are used to determine the increase of the number of cells in the sample. It is useful to evaluate the reactivity and the ability to function of the cells of an individual before and after having received a treatment.

It can also be used to distinguish the type of cell that actively proliferates by quantifying the number of cell divisions that they can operate on.

Finally it is a tool to be taken into account to determine the clinical evolution of a disease and its response to a given treatment . To identify and follow the cells during their proliferation process, several techniques can be used: a method consist in watching the loss of colour in the sample; a stain is added to the cells and loses a part of its colouration when the cell divides, in this case, the less colourful the sample, the more divided cells it contains.

Another indirect method consist in following the cell cycle by using marked proteins linked to the cell cycle; for example Ki-67, a nuclear antigen, is used as an early activation marker to identify the cells that are at the beginning of a new cell cycle because he is expressed only during active phases of the cell cycle (it is absent if the cell does not divide).

To determine the number of cells containing Ki-67, an antibody anti-ki-67, which is fluorescent, is added in the sample and sticks to ki-67, then the fluorescence is calculated, so the more fluorescent the sample, the more dividing cells it contains.

ATP concentration assays

ATP concentration assays are well-suited for high-throughput cell proliferation assays and screening. ATP, Adenosine Triphosphate, is the first source of energy of every living cell, therefore if a cell contains ATP, it means that the cell is alive.

To detect the presence of this nucleotide (component of DNA) luciferin and luciferase (a firefly enzyme and its substrate) are added to the sample. Luciferase uses the energy furnished by ATP to react with luciferin. The interest of this reaction is that it emits light. Then the luminescence is measured and the result is the following: the more luminescent the sample, the more living cells it contains.

DNA synthesis assays

DNA synthesis assays are the most accurate and reliable way to detect cell proliferation in the laboratory. DNA synthesis can be measured by adding Brdu (bromodeoxyuridine) that incorporates into newly replicated DNA, then a fluorescent anti-Brdu antibody is added to the sample and sticks to newly replicated DNA molecules; with this method, the more colourful the sample, the more newly created cells it contains.

It is possible to evaluate the rate of renewal of a given tissue, and to evaluate the duration of the cell cycle of the cells constituting this tissue. Such observations and manipulations can have multiple uses in medicine and cellular biology. You can use these tests to evaluate the impact and toxicity of a drug, to diagnose a disease, to study the evolution of a disease such as cancer or to predict a clinical outcome.



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