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 are non-coding genes? Give examples

Non-coding genes are segments of DNA that do not code for any protein but still play very important roles in the cell. These genes are transcribed into functional RNA molecules instead of mRNA. These RNAs do not get translated into proteins but perform regulatory, structural, or catalytic roles directly as RNA. Non-coding genes are a major part of the eukaryotic genome and help in many cellular processes such as gene regulation, RNA processing and maintaining genome stability. Many people think that only protein-coding genes matter, but non-coding genes also play a crucial role in regulating cellular activities. They help control when and how genes are turned on or off, assist in processing other RNAs and participate in building cell machinery like ribosomes and spliceosomes. There are several important types of non-coding genes, which can be grouped based on the RNA they produce and their functions. The main categories are: 1. rRNA Genes (Ribosomal RNA Genes) These genes produce ribos...

What are the differences between gene enhancers and gene silencers? How do enhancers and silencers regulate eukaryotic gene expression?

Enhancers and silencers are two important types of regulatory DNA sequences that play opposite roles in controlling gene expression in eukaryotic cells. They are non-coding DNA elements which do not produce proteins but control when, where and how much a gene is expressed. They work by interacting with transcription factors and RNA polymerase to either increase or decrease the level of transcription. Differences Between Gene Enhancers and Gene Silencers Gene enhancers and gene silencers are both regulatory DNA elements found in eukaryotic genomes. Their main role is to control the level of gene expression, but they work in opposite directions. The differences between them can be explained based on the following criteria: 1. Based on Function Enhancers increase the transcription of a gene. They make the gene more active and allow it to produce more RNA. On the other hand, silencers reduce or completely block transcription. They stop or decrease...

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...

What are epigenetic modifications? Give examples

Epigenetic modifications are heritable and reversible changes in gene expression that occur without altering the DNA sequence itself. These changes play a major role in how genes are turned on or off in different cells and at different times. Epigenetics helps explain how the same DNA sequence can produce different types of cells like skin cells, nerve cells and liver cells in the same organism. These modifications are very important during development, cellular differentiation, X-chromosome inactivation in females, genomic imprinting, aging and in many diseases such as cancer. There are the following three main types of epigenetic modifications: 1. DNA Methylation This is the most studied and well-understood form of epigenetic modification. In this process, a methyl group (–CH₃) is added to the cytosine base in DNA, mainly at CpG dinucleotides. These CpG regions are often found in clusters called CpG islands, which are located near gene promoters. DN...

What are the regulatory sequences of a typical eukaryotic gene? Give examples

In eukaryotic cells, gene expression is highly controlled by specific regulatory DNA sequences. These sequences do not code for proteins, but they decide when, where and how much a gene should be expressed. They mainly control the process of transcription, where RNA is made from DNA. A typical eukaryotic gene contains several important regulatory sequences, such as: Promoters Enhancers Silencers Insulators Response elements 1. Promoter The promoter is the main regulatory region of a gene. It lies just before the transcription start site, which is the point where RNA starts getting made. This is the site where RNA polymerase and transcription factors attach to begin transcription. One well-known part of the promoter is the TATA box, found around 25 to 35 base pairs before the start site. It helps RNA polymerase find the correct place to begin. Another element, the Initiator (Inr) sequence, is present near the +1 position and supports proper...

The Plum Pudding Model is one of the earliest models of atomic structure. It was built upon two major scientific contributions. The idea that atoms might contain both positive and negative charges was first proposed by William Thomson (commonly known as Lord Kelvin ) around the late 19th century. He suggested that positive and negative charges must be present together inside the atom to make it neutral. However, this idea remained a speculation until Sir Joseph John Thomson (Commonly known as J.J. Thomson ) made a major breakthrough in 1897 when he discovered a negatively charged particle, later called the electron, through his cathode ray tube experiments. He demonstrated that these rays were made of tiny, negatively charged particles that were much lighter than atoms. This was the first strong experimental evidence that atoms were divisible and had internal structure. After this discovery, in 1904, J.J. Thomson proposed a concrete atomic model to explain how these ele...

Describe what happens when a nonsense mutation is introduced into the gene encoding transposase within a transposon

Transposons, also known as jumping genes, are segments of DNA that can move from one position in the genome to another. This movement depends on a special enzyme called transposase, which is produced by a gene located inside the transposon itself. The transposase enzyme performs important functions like cutting the transposon from one place and helping it to insert into another. For the transposon to move, the transposase must be produced correctly and completely. When a nonsense mutation occurs in the transposase gene, it replaces a codon that normally codes for an amino acid with a stop codon. This causes the ribosome to stop protein synthesis early, resulting in a shortened and usually non-functional transposase enzyme. This leads to several important changes inside the cell. These changes are mainly: 1. Early Termination of Transposase Production The nonsense mutation introduces a premature stop codon in the transposase gene. Thi...

Describe the components of the promoter region of a eukaryotic gene

In eukaryotic genes, the promoter region is a special stretch of DNA that lies just before the gene. Its main job is to control when and where transcription starts. It does this by providing binding sites for RNA polymerase II and other transcription factors. The promoter region can be divided into two main parts: Core Promoter Proximal Promoter Elements Each of these parts has different types of DNA sequences that help start and regulate transcription. 1. Core Promoter This is the most essential part of the promoter and lies near the transcription start site (called the +1 position). It directly helps in assembling the transcription machinery. The core promoter usually includes the following elements: TATA Box: A short DNA sequence (TATAAA) usually located 25–35 base pairs upstream from +1 site. It helps in positioning RNA polymerase II. A special protein called TBP (TATA-binding protein) binds here to start the transcription complex. Initiator (Inr) Sequence: F...

M.Sc. (Zoology) (MSCZOO) Term-End Examination December, 2024 MZOE-001 : PARASITOLOGY Time : 2 Hours| Maximum Marks : 50 Note: (i) Attempt any five questions. (ii) All questions carry equal marks. 1. (a) What are endoparasites? Give any two examples. (2 Marks) Endoparasites are parasites that live inside the body of their host. They usually infect internal organs like the intestine, liver, lungs, or blood. These parasites depend on the host for nutrients and often cause harm by damaging tissues, stealing nutrients, or releasing toxins. They are commonly found in vertebrates, including humans and may be transmitted through contaminated food, water, or direct contact. Examples: 1. Taenia solium (pork tapeworm) – It lives in the human intestine and causes taeniasis. 2. Plasmodium vivax – A blood parasite that causes malaria in humans, transmitted by female Anopheles mosquitoes. (b) What is the difference between mechanical vector and biologic...

What are transcription factors? Describe the different categories of transcription factors

Transcription factors are special types of proteins that control the process of transcription in eukaryotic cells. Transcription is the first step of gene expression where the DNA is copied into RNA. These factors do not make RNA directly but help the enzyme RNA polymerase to start, stop or control the speed of this process. Transcription factors help the cell know when to turn a gene ON, when to turn it OFF and how much product the gene should make. They play a very important role in development, cell cycle, cell differentiation, stress response and hormone signaling. Transcription factors are mainly divided into two broad categories: Based on their function Based on their structure and DNA-binding domain 1. Types Based on Their Function There are the following two types: a) General Transcription Factors These transcription factors are needed for the transcription of almost all protein-coding genes. They help in forming the transcription initiation complex near the promoter...

Search This Blog

Study Paper Info