Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
TYPES OF SYNCHRONIZATION
(I)PHYSICAL CELL SEPARATION
(II)BLOCKADE
PHYSICAL Vs BLOCKADE SYNCHRONIZATION
CONCLUSION
REFFERENCE
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
Introduction.
Properties of Stem Cells.
Key Research events.
Embryonic Stem Cell.
Stem cell Cultivation.
Stem cells are central to three processes in an organism.
Research & Clinical Application of stem cell.
Research patents.
Conclusion.
Reference.
8. Biology and characterization of cultured cellsShailendra shera
Immediate environment and environment of surrounding medium governs the various properties of cell. The in vitro condition markedly affects the cellular property of cultured cells. For e.g. Reduction in Cell–cell and cell-material interaction. Therefore, it is imperative to develop understanding of biology of cells in response to various environmental conditions. Characterization of cells helps to identify the origin, purity and authenticity of cells and cell lines.
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
TYPES OF SYNCHRONIZATION
(I)PHYSICAL CELL SEPARATION
(II)BLOCKADE
PHYSICAL Vs BLOCKADE SYNCHRONIZATION
CONCLUSION
REFFERENCE
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
Introduction.
Properties of Stem Cells.
Key Research events.
Embryonic Stem Cell.
Stem cell Cultivation.
Stem cells are central to three processes in an organism.
Research & Clinical Application of stem cell.
Research patents.
Conclusion.
Reference.
8. Biology and characterization of cultured cellsShailendra shera
Immediate environment and environment of surrounding medium governs the various properties of cell. The in vitro condition markedly affects the cellular property of cultured cells. For e.g. Reduction in Cell–cell and cell-material interaction. Therefore, it is imperative to develop understanding of biology of cells in response to various environmental conditions. Characterization of cells helps to identify the origin, purity and authenticity of cells and cell lines.
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Role of serum and supplements in culture medium k.skailash saini
ROLE OF SERUM AND SUPPLEMENTS IN CULTURE MEDIA
Serum is a complex mix of albumins, growth factors and growth inhibitors.
Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture.
Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells.
Calf serum is used in contact-inhibition studies because of its lower growth-promoting properties.
Normal growth media often contain 2-10% of serum.
Supplementation of media with serum serves the following functions :
Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells.
Serum provides several growth factors and hormones involved in growth promotion and specialized cell function.
It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide.
It provides protease inhibitors which protect cells from proteolysis.
It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc.
It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
It also acts a buffer.
Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex.
Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks .
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
This presentation contains all the material regarding History of animal cell culture and different methods of organ and tissue culture.Hope it will be helpful..
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Role of serum and supplements in culture medium k.skailash saini
ROLE OF SERUM AND SUPPLEMENTS IN CULTURE MEDIA
Serum is a complex mix of albumins, growth factors and growth inhibitors.
Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture.
Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells.
Calf serum is used in contact-inhibition studies because of its lower growth-promoting properties.
Normal growth media often contain 2-10% of serum.
Supplementation of media with serum serves the following functions :
Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells.
Serum provides several growth factors and hormones involved in growth promotion and specialized cell function.
It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide.
It provides protease inhibitors which protect cells from proteolysis.
It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc.
It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
It also acts a buffer.
Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex.
Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks .
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
This presentation contains all the material regarding History of animal cell culture and different methods of organ and tissue culture.Hope it will be helpful..
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to duplication of its DNA (DNA replication) and division of cytoplasm and organelles to produce two daughter cells.
This presentation is regarding the normal cell cycle through which a cell passes throughout its life. It highlights each step in the formation of daughter cells from a mother cell. It puts light on the events in both the interphase and division (mitotic) phase and the resting (G0 phase).
You will also get knowledge about the cell cycle checkpoints and the cellular brakes, the proteins that keeps the cell to divide normally, and how the abnormalities in these proteins results in defects of cell cycle and subsequently leads to uncontrolled cell division and cancer formation.
This presentation consists of topics related to oncogene, proto oncogene, Tumor suppresor gene, Ras gene family and structure and functions of tumor suppressor gene.
Stem cells are the cells which have the capability to differentiate into any cells of the body when provided with right stimulus and environment. This presentation teaches about stem cells, characteristics, types and cultivation of stem cells in artificial environment. Sample practice questions are also provided in the end to review the concept learned from this presentation.
Workplace safety is an important aspect to protect personnel against injury or serious accident.In case of animal cell culture safety takes a front seat due to nature of work i.e. handling of human cells and tissues, viruses with high potential to cause infections to humans and other adventitious micro organisms. This presentation presents various methods of safety to protect lab personnel from infectious biological agents.
Media is one of the important components for in vitro cultivation of animal cells. Every animal cells have specific requirements and media are designed by keeping in mind those requirements. However, the basic components and design principle remains the same. Every cell culture media contain carbon source, nitrogen source, trace elements, pH indicator, antibiotics ( although it is not recommended) for high value cell culture applications. While designing media various aspects are considered such as availability, cost effectiveness, types off cells to be grown and regulatory requirements. Tis slide also contains sample MCQs questions
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
This slide explains the various basic aspect of animal cell culture, cell line and cell strain, initiation and maintenance of primary cell culture, characteristic of primary cell culture and their applications. It also contains MCQs for practice.
This presentation presents an overview of definition, equipment, various cell culturing methods,
characterization, and applications of animal cell culture. This presentation also contains MCQs to acquaint reader about types of question asked in various competitive examinations.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
2. LT.12 Cell Synchronization & Transformation
Content Outline
1. Cell synchronization
2. Methods of cell synchronization
3. Cell synchronization using thymine double block method
4. Confirmation of cell synchronization
5. Transformation ( Cellular)
6. Immortalization
7. Mechanism of immortalization
8. Practice questions
3. Cell synchronization
•Cell synchronization is a process by which cells at different stages of the cell cycle in a culture are
brought to the same phase.
•Through synchronization, cells at distinct cell cycle stage could be obtained.
•Many cell-synchronization protocols involve placing cells under growth inhibitory conditions for
periods of time related to the duration of specific cell-cycle phases.
•Cell synchronisation is used to study the progression of cells through the cell cycle
Cell can be synchronized in any of the following main stages of cell cycle
G0/G1 phase – cell rest and recovery in preparation for subsequent rounds of cell division
S Phase – DNA replication (interphase)
G2/M phase – chromosome segregation and mitosis
Targeting the cell cycle stage:
• Release from GO arrest,
•release from M- and S-phase blocking agents,
• mitotic detachment
•centrifugal elutriation
4. Methods of cell synchronization
Cell
synchronization
Physical
Centrifugal
treatment
Flow Cytometry
Affinity of
antibodies
Chemical
Double
thymidine block
Taxol arrested
cells with Cdk
inhibitors
Nocodazole
treatment
5. Physical Fractionation
•Physical fractionation or cell separation techniques can be based on cell density, cell size,
affinity of antibodies on cell surface epitopes and light scatter or fluorescent emission by labeled
cells.
•Centrifugal separation or fluoresecene-activated cell sorting are primarily used for physical
fractionation. Centrifugal separation enables the separation of cells based on size and
sedimentation velocity. Fluorescence-activated cell sorting (FACS) sorts cells on based on
differences which can be detected by light scatter (e.g. cell size) or fluorescence emission (by
penetrated DNA, RNA, proteins, antigens). This can be carried out using a flow cytometer or
fluorescence-activated cell sorter.
Physical methods
6. Chemical methods
Chemical Blockade
•As the names suggests this method uses chemicals such as Thymidine to block metabolic
reactions. This can be achieved through inhibition of DNA synthesis largely during the S-Phase o
the cell cycle. Inhibitors such as thymidine, aminopterin, hydroxyurea and cytosine arabinoside can
have variable effects. Serum starving your cells for 24 hrs will result in an accumulation of cells at G1
phase. The effects of serum starvation can be reversed by the addition of serum to media once cell
synchrony has occured.
•Different chemicals can induce cell synchronisation at different stages in the cell cycle
Cell Cycle Stage Targeted Treatment used
G1 arrest Double Thymidine block,Serum starvation. Inhibition of cyclin dependent
kinase (CDKs)
G2 arrest Inhibition of microtubules, Inhibition of cyclin dependent kinase (CDKs)
M-Phase Taxol, Nocodazole
7. •To synchronise cells at the G1/S border a freshly prepared thymidine solution (16 mM) was made
up in complete medium and filter-sterilised using a 0.22 µm filter disc.
•Cells (1 X 107) were cultured in 60 ml complete medium containing thymidine (2 mM) for 16 h in a
175 cm3cell culture flask.
•The cells were then resuspended in complete medium (52.5 ml) for 8 h to allow cells to
• reenter the cell cycle.
•Cells were harvested by centrifugation at 400 x g for 5 min and washed X 2 in complete medium
(10 ml). Cells (1 X 107) were cultured in 60 ml complete medium containing thymidine (2mM) for 16
h to synchronise cells at the G1/S border.
•Cell synchronisation was confirmed by flow cytometry.
Cell synchronization using double thymidine block
Protocol adopted as it is from
https://www.elisagenie.com/cell-synchronisation-methods/
Also visit this site for other detailed protocol
8. •Cell synchronisation can be confirmed by microscopy or flow cytometry. Microscopy allows you to
see what is actually going on inside you cells.
•Flow cytometry enables you to compare your treated synchronised cells against a asynchronous
control. Briefly the protocol is as follows;
1. Fix and permeabilize your cells in 70 % ethanol
2. Stain with 40 µg/ml propidium iodide, and include 25 µg/ml of Rnase (to degrade RNA and
ensure that you stain DNA only).
3. Run your samples on the flow cytometer.
Confirmation of cell synchronization
9. Transformation ( Cellular transformation)
•Transformation of cultured cells implies a spontaneous or induced permanent phenotypic change
resulting from a heritable change in DNA and gene expression.
•Although transformation can arise from infection with a transforming virus, such as polyoma, or
from transfection with genes such as mutant ras, it can also arise spontaneously, after exposure to
ionizing radiation, or after treatment with chemical carcinogens
•Transformation is associated with genetic instability
Phenotypic changes associated with transformation are:
i. Immortalization
ii. Aberrant growth control
iii. Malignancy
Characteristic of Transformed cells
Genetic instability:
• Cells have high rate of undergoing mutations and changes their genetic make up even with
slight induction
• Continuous cell lines, particularly from tumors of all species, are very unstable.
• Deletion or alteration in DNA surveillance genes, such as p53, are usually implicated.
10. •Continuous cell lines are usually heteroploid, meaning they show a wide range in chromosome
number among individual cells in the population, implying substantial genetic diversity.
There are two main causes of genetic heterogeneity:
(1) the spontaneous mutation rate appears to be higher in vitro, associated, perhaps, with the high
rate of cell proliferation and defective DNA surveillance genes, particularly p53,
(2) mutant cells are not eliminated unless their growth capacity is impaired.
Chromosomal aberration
•Aberration refers to a characteristic that deviates from the normal type
•Chromosomal abnormalities occur when there is either an excess or a loss of a total chromosome
or when a portion of a chromosome is absent (macrodeletions and microdeletions).
•Most tumor culture show signs of aneuploidy, meaning deviations from the normal complement of
chromosomes. Some specific aberrations are associated with particular types of malignancy
As result of chromosomal aberrations, the transformed cells show anchorage independence, loss
of contact inhibition, and low serum requirement.
11. Anchorage independence
There occur several changes on the cell surfaces of transformed cells. These include alterations in
the cell surface glycoproteins and integrin’s, and loss of fibronectin. Some of the transformed cells
may totally lack cell adhesion molecules (CAMs). The modifications on the surface of transformed
cells leads to a decrease in cell—cell, and cell- substrate adhesion.
Contact inhibition:
The transformed cells are characterized by loss of contact inhibition. This can be observed by the
morphological changes in the disoriented and disorganized monolayer cells. This result in a
reduced density limitation of growth, consequently leading to higher saturation density compared to
normal cells.
Low serum requirement:
In general, transformed cells or tumor cells have lower serum dependence than the normal cells.
This is mostly due to the secretion of autocrine growth factors by the transformed cells
12. •Most normal cells have a finite life span of 20 to 100 generations, but some cells, notably those
from rodents and from most tumors, can produce continuous cell lines with an infinite life span.
•These dominantly acting genes synthesize products which inhibit the cell cycle progression.
•It is strongly believed that immortalization occurs due to inactivation of some of the cell cycle
regulatory genes e.g. Rb, p53 genes.
•Out of 10 genes involved in senescence, some of gene s owing to mutation negatively regulate
the expression of telomerase, required for the terminal synthesis of telomeric DNA, which
otherwise becomes progressively shorter during a finite life span, until the chromosomal DNA can
no longer replicate.
Immortalization of cell
13. Mechanism of Immortalization
It has been assumed that immortalization is a multistep process involving the inactivation of a
number of cell cycle regulatory genes, such as Rb and p53. The SV40 LT gene is often used to
induce immortalization. The product of this gene, T antigen, is known to bind Rb and p53. By doing
so, it not only allows an extended proliferative life span but also restricts the DNA surveillance
activity of genes like p53, thereby allowing an increase in genomic instability and an increased
chance of generating further mutations favorable to immortalization (e.g., the upregulation of
telomerase or the downregulation of one of the telomerase inhibitors). Transfection of the
telomerase gene with a regulatable promoter is sufficient to immortalize cells
Methods of Immortalization: Making cells immortal
Several methods exist for immortalizing mammalian cells in culture conditions.
With years of experience in cell immortalization. Recombinant lentiviral, retroviral (MMLV) and
adenoviral viruses expressing EBV, HPV-16 E6/7 and SV40 T antigens, hTERT, p53 and RB
siRNAs, and ras & myc mutants. All these tools will make your cell immortalization project simpler
and easier than ever before.
Follow link for process of immortalization: https://www.biocat.com/cell-biology/cell-immortalization
14. Test your Understanding
1.Cellular Transformation refers to
a. Transforming cells by inserting exogenous DNA
b. Transforming cells through alteration of endogenous DNA
c. Both (a) and (b)
d. None of the above
2.Centrifugal elutriation is used for
a. Cell synchronization
b. Sorting of cells
c. Both (a) and (b)
d. None of the above
3.What happens to cells if p53 and Rb genes are inactivated
a. Cells dies instantaneously
b. Cell undergo apoptosis
c. Cell becomes immortal
d. None of the above
4.Anchorage independency is the characteristics of……………………..cells.
5. Starvation of cells of nutrient is one method of synchronizing cellular growth in culture. ( True / False)
*Questions adapted from: Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
15. References
1. https://www.elisagenie.com/cell-synchronisation-methods/
2. https://link.springer.com/protocol/10.1007%2F978-1-4939-6603-5_1
3. https://experiments.springernature.com/articles/10.1007/978-1-4939-6603-5_1
4. https://www.sciencedirect.com/science/article/pii/S0091679X08615824
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156087/
6. https://info.abmgood.com/blog/your-basic-guide-to-cell-line-immortalization
7. https://www.biocat.com/cell-biology/cell-immortalization
MCQ Practice questions
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
Available on: amazon.com