What is the principle of LDH assay for the measurement of cell death?

Get link
Facebook
X
Pinterest
Email
Other Apps

LDH assay is one of the most commonly used biochemical methods to measure cell membrane damage and cell death, especially during necrosis or late-stage apoptosis. LDH stands for Lactate Dehydrogenase, which is an intracellular enzyme found in the cytoplasm of almost all cells. Under normal conditions, LDH remains inside the cell. But when the cell membrane becomes damaged or ruptured due to stress, toxin, or cell death, LDH leaks out into the surrounding medium. The LDH assay takes advantage of this leakage to estimate the extent of cell death.

Principle of the LDH Assay

The principle of the LDH assay is based on the ability of LDH enzyme to catalyze a reaction that changes lactate to pyruvate. This reaction also results in the conversion of NAD⁺ to NADH. The NADH produced can then react with specific substrates to create a colored product, which can be measured using a spectrophotometer.

The overall reaction looks like this:

Lactate + NAD⁺ → Pyruvate + NADH + H⁺

The NADH produced is responsible for reducing a tetrazolium salt or INT reagent in the assay. This reduction results in a color change and the intensity of the color correlates with the amount of LDH released.

The more LDH in the culture medium, the more NADH is formed, leading to more intense color production. This color is measured at a specific wavelength (usually 490 nm or 540 nm) and the resulting absorbance value indicates the level of cell death.

Importance and Use of LDH Assay

It is used to measure cytotoxicity in drug testing and cell viability studies.

It helps to distinguish between necrotic and apoptotic cell death.

It is non-radioactive, easy to perform and suitable for high-throughput experiments.

It does not require destruction of the remaining cells, so the same sample can be used for other tests.

Get link
Facebook
X
Pinterest
Email
Other Apps

Comments

What is the difference between the classical concept and the modern concept of genes?

The concept of the gene has evolved from a simple unit of heredity to a complex molecular entity. During Mendel's time, genes were understood only through the inheritance of physical traits. This formed the classical concept. With the discovery of DNA, its double helix structure and the development of molecular biology, the gene is now defined by its chemical structure and functional properties. This is known as the modern concept. For understanding the difference between the classical and modern concept of genes, the following criteria are used: 1. Based on Definition or Concept Classical Concept: Gene was defined as an abstract unit of heredity responsible for controlling a single trait. It was known through breeding experiments and inheritance patterns. Modern Concept: A gene is a segment of DNA that contains the information to produce a functional product, either a protein or an RNA. It is both a structural and functional unit of...

What is gene mapping? How do the linked genes help in gene mapping?

Gene mapping is the method used to determine the location of genes on a chromosome and the distance between them. It helps in identifying the exact position of a gene responsible for a particular trait or disease. The concept started with the work of Thomas Hunt Morgan in the early 1900s when he studied Drosophila melanogaster (fruit fly) and observed that some traits are inherited together. This was because the genes responsible for those traits were located close to each other on the same chromosome. This phenomenon is known as linkage. There are two main types of gene mapping: 1. Genetic Mapping (Linkage Mapping): Genetic mapping uses the frequency of recombination or crossing over between genes to estimate their distance on a chromosome. It gives a relative position of genes rather than their exact physical location. 2. Physical Mapping Physical mapping uses molecular biology techniques to determine the exact nucleotide sequence of DNA and the exact physical ...

The question of which molecule can be considered the "first living molecule" is complex and doesn't have a definitive answer. This depends on whether we are discussing the molecule that played a key role in the origin of life or the first organic molecules that formed chemically on early Earth. While RNA is considered crucial in the emergence of life, amino acids were likely the first molecules to appear in Earth's primordial environment. Understanding these distinctions clarifies the discussion about whether RNA or amino acids came first. Introduction: 1. According to the Origin of Life: In the context of the origin of life, the first molecule is likely RNA. RNA's ability to store genetic information and catalyze its own replication (acting as both a genetic material and an enzyme) gives it a key role in early life. The RNA World Hypothesis suggests that RNA existed before amino acids or proteins, as it could facilitat...

Why is the frequency of double crossover overly low?

The frequency of double crossover is usually much lower than expected. This is mainly due to a natural genetic mechanism called interference, which controls the distribution of crossover events during meiosis. Crossover is essential for genetic recombination but is also tightly regulated to prevent instability in the genome. The lower frequency of double crossovers can be explained by the following reasons: 1. Physical Constraints of Chromosomes Chromosomes have a limited length and physical structure. When a crossover happens at one region of a chromosome, the local chromatin structure and spatial arrangement become less favorable for another crossover nearby. This physical limitation reduces the chance of two crossovers occurring very close to each other on the same chromosome segment. 2. Crossover Interference One of the main reasons for reduced double crossovers is the phenomenon called interference. Interference is the effect where the occurrence of one crossover decrea...

What is the difference between regulatory gene and structural gene?

Genes are basic units of heredity that carry instructions for the development and functioning of living organisms. Among various types of genes, regulatory genes and structural genes are essential for the proper functioning of cells, but their roles are quite different. Regulatory genes mainly control other genes, while structural genes directly code for proteins that form the body's structures or perform specific functions. To understand their differences clearly, we will compare them based on important criteria. 1. Based on Function The main function of regulatory genes is to control or regulate the expression of other genes. They produce regulatory proteins, such as repressors or activators, that influence whether structural genes are switched on or off. This control mechanism ensures that genes are expressed only when needed. In contrast, structural genes code directly for proteins or RNA molecules that are involved in building cellula...

Atoms are the basic units of matter and are made up of smaller components called subatomic particles. There are many types of subatomic particles known to science, but in the context of basic atomic structure, only three are considered most important: electrons, protons and neutrons. These three particles differ in their location, charge and mass. Together, electrons, protons and neutrons form the complete structure of atoms. Their arrangement and interaction define the atom's properties, chemical behavior, and participation in physical and chemical processes. These subatomic particles laid the foundation of modern atomic theory and quantum chemistry. 1. Electron Electrons are negatively charged subatomic particles. They are extremely small in mass and are found outside the nucleus of the atom in specific regions called orbitals or shells. The charge of an electron is −1 and its mass is approximately 1/1836 of a proton or neutron, which is around 9.1 × 10⁻³¹ kg, meaning it is n...

Differentiate between linked genes and unlinked genes?

Genes are specific sequences of DNA that code for proteins and determine traits in an organism. During the study of chromosomal theory of inheritance, scientists found that not all genes behave the same way. Some genes tend to be inherited together while others assort independently. Based on this behavior, genes are divided into two types: linked genes and unlinked genes. This concept is very important in genetics because it helps in understanding how traits are passed on and how gene positions can be mapped on chromosomes. These differences are explained based on specific criteria: 1. Based on Chromosomal Location Linked genes are located close to each other on the same chromosome. Because of their close physical proximity, they usually move together during meiosis and are inherited as a group. For example: In Drosophila melanogaster (fruit fly), the genes for eye color and wing shape are located close to each other on the X chromosome. Unlinked genes are ...

Define and distinguish sex-linked, sex-limited and sex-influenced characters

In genetics, traits can be influenced or expressed differently depending on the sex of the individual. Some traits are linked to sex chromosomes, while others are affected by hormonal or physiological differences between males and females. To describe these traits more precisely, geneticists use three main terms: sex-linked, sex-limited and sex-influenced traits. Although these terms may sound similar, they refer to different types of genetic expression related to sex. Understanding the distinction among these three is important for grasping how certain traits are inherited and expressed differently in males and females. 1. Sex-linked characters: These are traits controlled by genes that are located on the sex chromosomes, usually on the X chromosome in humans. Because males have only one X chromosome (XY) and females have two (XX), the pattern of inheritance and expression is different in both sexes. Most sex-linked traits are X-linked, and very few are Y-linked...

Give two examples of gene interaction resulting in the formation of structural proteins

Gene interaction refers to a situation where two or more genes influence the same trait. In the case of structural proteins, sometimes the final functional protein is not made from a single gene product but is the result of the combination of different polypeptides produced by different genes. Such interaction is especially important in the formation of complex structural proteins that require the association of multiple chains to become functional. Two good examples of this kind of gene interaction are seen in haemoglobin and MHC (Major Histocompatibility Complex) molecules. 1. Haemoglobin (HbA) Haemoglobin is the oxygen-carrying protein found in red blood cells. The adult type of haemoglobin, called HbA, is a tetramer made up of two alpha-globin chains and two beta-globin chains. These chains are coded by different genes: The alpha-globin gene is located on chromosome 16. The beta-globin gene is located on chromosome 11. Both these...

How is recessive epistasis different from duplicate recessive epistasis?

In classical Mendelian genetics, one gene controls one trait. But in real life, many traits are controlled by two or more genes and sometimes one gene can affect or interfere with the expression of another gene. This interaction is called epistasis. Here, we will compare two types: recessive epistasis and duplicate recessive epistasis. Both involve two genes, but the way they interact is different. Recessive Epistasis Recessive epistasis occurs when the recessive alleles of one gene (in homozygous form) can mask or suppress the effect of another gene. In this case, the second gene can be dominant or recessive, but its expression will not appear if the first gene is homozygous recessive. This means one gene (called the epistatic gene) is stronger and can block the phenotype controlled by the second gene (called the hypostatic gene), but only when present in recessive form. Example: A good example is coat colour in mice. One gene (A) controls pigment production. If...

Search This Blog

Study Paper Info