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.
Biology and characterization of the cell cultureKAUSHAL SAHU
Introduction
History
Important terminology
Biology of culture cell
Characterization of culture cell
Application of animal culture
Conclusion
References
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
Biology and characterization of the cell cultureKAUSHAL SAHU
Introduction
History
Important terminology
Biology of culture cell
Characterization of culture cell
Application of animal culture
Conclusion
References
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
This presentation is about the basics of cytotoxicity and the possible cellular fates a cell goes through and cellular and molecular level alterations. The nanoparticle related cytotoxicity and its emerging impacts in the environment and health and the disease caused by it is also discussed briefly.
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.
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
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..
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
This presentation is about different staining methods to distinguish between a viable and a non-viable cell. These staining techniques are of immense importance to study different diseased cells such as cancer cells, nerve cells to evaluate the respiratory and metabolic activity of a cell and the potential effects of various drugs to kill the diseased cell.
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
This presentation is about the basics of cytotoxicity and the possible cellular fates a cell goes through and cellular and molecular level alterations. The nanoparticle related cytotoxicity and its emerging impacts in the environment and health and the disease caused by it is also discussed briefly.
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.
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
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..
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
This presentation is about different staining methods to distinguish between a viable and a non-viable cell. These staining techniques are of immense importance to study different diseased cells such as cancer cells, nerve cells to evaluate the respiratory and metabolic activity of a cell and the potential effects of various drugs to kill the diseased cell.
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
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
The Main Advantage
The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher and slower pace.
Aquatic research
In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains.
This research started in 1981 when Clarice Yentsch used flow cytometry to measure the fluorescence in a red tide producing dinoflagellates
Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity, to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity,and to measure biogeochemical rates of multiple processes in the ocean
Cell Proliferation assay
Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments
Cell counting
Cell sorting
Determining cell characteristics and function
Detecting microorganisms
Biomarker detection
Protein engineering detection
Diagnosis of health disorders such as blood cancers
Flow cytometry can be used for cell cycle analysis to estimate the percentages of a cell population in the different phases of the cell cycle, or it can be used with other reagents to analyze just the S phase.
Why flow cytometry is ideal for cell cycle analysis
Live-cell cycle analysis stains—Vybrant DyeCycle stains
Classic DNA cell cycle stains such as Hoechst 33342 and DRAQ5 for cell cycle analysis, but most of these have limitations that have to be considered when using them in an experiment which is why the Invitrogen Vybrant DyeCycle stains for live-cell cycle analysis were developed.
Fixed-cell cycle analysis stains FxCycle reagents
We offer classic DNA cell cycle stains such as DAPI, PI, and 7-AAD for fixed cell cycle analysis, but these reagents do not cover the full spectrum of laser excitation available.
The FxCycle reagents offer options for the 405 nm (violet) and 633 nm (red) laser thereby increasing the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses such as immunophenotyping, apoptosis analysis, and dead cell discrimination.
Precise—Accurate cell cycle analysis in living cells
Safe—Low cytotoxicity for combining with additional live cell experiments
Cell sort compatible—Easily sort cells based on phase of the cell cycle
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.
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.
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
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
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.
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
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.
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.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
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.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
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
2. Measurement of viability and cytotoxicity
Outline
1. Definition of Viability & Cytotoxicity
2. Methods of assessment
a) Dye Exclusion methods
b) Colorimetric Assay
c) Enzyme release assay
d) Fluorometric assay
e) Luminometric assay
3. Practice Questions
3. Definition: Viability and Cytotoxicity
Cells in culture should be routinely checked for their health status to ascertain their normal
physiological growth. There are various methods to ascertain the health status of growing cells.
These are based on various cell functions such as enzyme activity, cell membrane permeability, cell
adherence, ATP production, co-enzyme production, and nucleotide uptake activity. Before going
further, let’s start with definition:
What is viability?
Viability of the cells represents the capability of their existence, survival and developments.
What is cytotoxicity?
Chemicals with a capability to destroy cells i.e toxic/ harmful to cells causing either cells death or
deviation from normal physiological behavior.
4. Methods of Assessment
Cell viability and cytotoxicity assays are based on colorimetric, fluorometric and bioluminescent
detection chemistries. The tree below shows various modes of assessing viability and
cytotoxicity.
Cell viability and
cytotoxicty
Dye exclusion
method
Colorimetric
assay
Fluorometric
assay
Luminometric
assay
5. Dye exclusion methods
This techniques is based on exclusion of dye by viable cells.
Dead cells have damaged plasma membrane and increased permeability to dyes. Therefore, Viable cells remains
unstained whereas dead cells are stained.
Common dyes:
Trypan blue: It selectively stains dead cells. Staining with trypan blue followed by microscopic examination on
hemocytometer is frequently used method to determine the cell number and percent viability in a population of
cells.
Dead cells are stained blue
Erythrosine B : Erythrosine B, also known as erythrosine or Red No. 3 is a vital stain.
It also has fluorescent capacity.
The principle is similar to Trypan Blue. It asses memrane integrity and penetrates plasma membrane of dead
cells.
Fluorescein diacetate (FDA): Fluorescein diacetate (FDA) is a non-fluorescent molecule, which is hydrolysed to
fluorescent fluorescein in live cells.
It is also a vital stains.
The hydrolysis is performed by esterase enzymes present in viable cells only.
viable cells retain the dye and fluoresce whereas nonviable cells do not. Fluorescein diacetate is not cytotoxic.
6. Colorimetric assay
Colorimetric assays: MTT assay, MTS assay, XTT assay, WST-1 assay, WST-8 assay, LDH assay, SRB assay,
NRU assay and crystal violet assay.
Principle:
•It is the measurement of a biochemical marker to evaluate metabolic activity of the cells.
•Reagents used in colorimetric assays develop a color in response to the viability of cells, allowing the colorimetric
measurement of cell viability via spectrophotometer
MTT assay
The MTT assay is colorimetric assay which uses reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide, or MTT.
It is based on the ability of mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent
cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple
color.
Formazan crystals are then dissolved using a solubilizing solution and absorbance is measured at 500-600
nanometers using a plate-reader.
The darker the solution, the greater the number of viable, metabolically active cells.
During spectrophotometric determination, the absorbance value will increase with time. % viability is calcultaed
as:
7. XTT Assay
It is based on the reduction of a yellow tetrazolium salt (sodium 3´-[1- (phenylaminocarbonyl)- 3,4-
tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate or XTT) to an orange formazan
dye by metabolically active cells
XTT is simply for measuring proliferation and is therefore an excellent solution for quantitating cells
and determining their viability.
The XTT assay is used to measure cellular metabolic activity as an indicator of cell viability,
proliferation and cytotoxicity.
It is used for the measurement of cell proliferation in response to growth factors, cytokines and
nutrients
https://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/cell-proliferation-kit-xtt-assay.html
8. Enzyme release assays:
The membrane integrity of cells can also be assessed by estimating the enzymes released.
Lactate dehydrogenase (LDH) has been the most widely used enzyme for this purpose.
The damaged / non viable cells with compromised membrane integrity releases lactate
dehydrogenase. LDH converts Lactate to pyruvate, generating NADH from NAD. The NADH
interacts with the probe to generate color at 450 nm.
The amount of colored product formed is directly proportional to the LDH activity in the sample.
The LDH activity is determined as NADH oxidation or INT reduction over a defined time period.
The resulting red formazan absorbs maximally at 492 nm and can be measured quantitatively at
490 nm.
9. Fluorometric assay
Fluorometric assays: alamarBlue assay and CFDA-AM assay.
Fluorometric assays of cell viability and cytotoxicity are easy to perform with the use of a
fluorescence microscope, fluorometer, fluorescence microplate reader or flow cytometer.
Fluorometric assays are also applicable for adherent or suspended cell lines and easy to use.
Alamar Blue
AlamarBlue Cell Viability Reagent is an indigo-colored, non-toxic reagent that detects
metabolically active cells and is used for the quantitative analysis of cell viability and proliferation
Resazurin, the active ingredient of alamar Blue reagent upon uptake by living cells is reduced to
resorufin, a compound that is red in color and highly fluorescent.
Absorbance- or fluorescence-based plate reader are used to assess change in viability.
Absorbance (detected at 570 and 600 nm) or fluorescence (using an excitation between 530–
560 and an emission at 590 nm).
Add alamar blue
solution to complete
media directly
Incubate
1-4 hours
Spectrophotometric/
Fluorometric
observation
10. Luminometric assay
Luminometric assays: ATP assay and real-time viability assay.
ATP assay
•When cells damaged lethally and lose membrane integrity, they lose the ability to synthetize
ATP and the ATP level of cells decreases dramatically.
•The ATP assay is based on the reaction of luciferin to oxyluciferin. Enzyme luciferase catalyzes
this reaction in the presence of Mg2+ ions and ATP yielding a luminescent signal.
•There is a linear relationship between the intensity of luminescent signal and ATP concentration
or cell number
11. 1. MTT assay is a type of
a. Colorimetric assay
b. Fluorometric assay
c. Emzyme release assay
d. None of the above
2. What is the property of vital stain?
a. It should kill cells in culture on addition
b. It should be non toxic to cells in culture
c. Both (a) and (b)
d. None of the above
3. Dye exclusion method assays
a. Membrane integrity of cell
b. Enzyme reduction capability.
c. Integrity of DNA
d. None of the above
4. ATP assay is an example of
a. Luminometric method
b. Colorimetric method
c. Enzyme release
d. DNA binding
5. Fluorescein diacetate (FDA) is hydrolyzed by cellular esterase to …………
a. Resazurin
b. Fluorescein
c. Formazan
d. Dimethyl sulphoxide
*Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
Practice questions: MCQs*