The document summarizes various methods used to measure apoptosis and cell viability, including membrane integrity assays, functional assays, DNA labeling assays, and morphological assays. Membrane integrity assays like trypan blue exclusion, annexin V binding, and LDH leakage determine cell viability based on plasma membrane integrity. Functional assays like MTT, XTT, and Alamar Blue measure cell metabolism. DNA labeling assays use fluorescent conjugates to label cells, while morphological assays examine changes in cell morphology during apoptosis. The document provides details on the principles, materials, and procedures for several common assays used to measure apoptosis and cell viability.
Creative Bioarray provides Cell Apoptosis Assays to all of our customers. The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms.
https://www.creative-bioarray.com/cell-apoptosis-assays.htm
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
Cell proliferation assays are used to monitor the dynamic growth of a cell population or to detect daughter cells in a growing population.
On the other hand, cell viability assays assess how healthy the cells are by measuring markers of cellular activity.
https://www.creative-bioarray.com/products/cell-viability,-proliferation-and-cytotoxicity-list-226.htm
Creative Bioarray provides Cell Apoptosis Assays to all of our customers. The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms.
https://www.creative-bioarray.com/cell-apoptosis-assays.htm
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
Cell proliferation assays are used to monitor the dynamic growth of a cell population or to detect daughter cells in a growing population.
On the other hand, cell viability assays assess how healthy the cells are by measuring markers of cellular activity.
https://www.creative-bioarray.com/products/cell-viability,-proliferation-and-cytotoxicity-list-226.htm
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells
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.
Nowadays, Cell and Molecule Analysis are critical steps for studying cellular mechanisms in any Life Sciences lab, as it offers additional information about Intracellular Molecular Targets in a real Biological environment.
Canvax™ offers a wide range high quality of Cell-based tools and Molecule Detection Kits designed for greater accuracy and increased Throughput for most common measurements like Cell Viability, Proliferation, Cytotoxicity, Reporter Gene or Oxidative Stress Assays.
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
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.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
6. Features distinguishing live from dead cells include the loss
of transport function across plasma membrane which results
from loss of membrane integrity.
Cells must be counted within 3-5 min because the number of
blue-staining cells increases with time after addition of the
dye.
Large numbers of samples have to be counted, it may be
inconvenient to perform all the tests on the same day by
counting one cell suspension at a time before staining the next
sample.
• - Viable cells : small, round and refractive
• - Non-viable cells : swollen, larger, dark blue
Exclusion dyes
7. Ethidium bromide (EtBr) and propidium iodide (PI)
PI binds to nucleic acids upon membrane damage :
flow cytometric techniques depend on fluorescence, PI
is ideally suitable for the rapid evaluation of the
permeability properties of large numbers of cells while
maintaining good statistical accuracy.
PI is impermeable to intact plasma membrane.
Intercalates with DNA or RNA red
Fluorescent dyes
8. Fluorescein diacetate (FDA) is a nonpolar ester which passes
through plasma membranes and is hydrolyzed by intracellular
esterases to produce free fluorescein, the polar fluorescein is
confined within cells which have an intact plasma membrane and
can be observed under appropriate excitation conditions.
Undamaged cell : highly fluorescent fluorescein dye
Damaged cell : fluoresce only weakly
greenish-yellow at 450-480 nm
9. Intact cell –
PI and FDA is added
Fluorescein in
intact cells
Schematic illustration of the principle of
PI/FDA cell viability assay
● FDA (Fluorescein diacetate)
● PI (Propidium iodide)
Plasma membrane is damaged
; fluorescein leaks out
PI enters and strains
nucleic acids
10. • Annexin V: An Early Marker of Apoptosis
•
One of the earliest indications of apoptosis is the translocation of the
membrane phospholipid phosphatidylserine (PS) from the inner to the
outer leaflet of the plasma membrane.
• Once exposed to the extracellular environment, binding sites on PS
become available for Annexin V, a 35-36 kDa, Ca 2+-dependent,
phospholipid binding protein with a high affinity for PS.
• The translocation of PS precedes other apoptotic processes such as
loss of plasma membrane integrity, DNA fragmentation, and chromatin
condensation.
• As such, Annexin V can be conjugated to biotin or to a fluorochrome
such as FITC, PE, APC, Cy5, or Cy5.5, and used for the easy, flow
cytometric identification of cells in the early stages of apoptosis
11. • Because PS translocation also occurs during necrosis, Annexin V is not an
absolute marker of apoptosis.
• Therefore, it is often used in conjunction with vital dyes such as 7-amino-
actinomysin (7-AAD) or propidium iodide (PI), which bind to nucleic acids,
but can only penetrate the plasma membrane when membrane integrity is
breached, as occurs in the later stages of apoptosis or in necrosis
• Result
• annexin-/PI-, annexin +/PI-, annexin+/PI+ and annexin –/PI+
12. • No Apoptosis = Cell Viability
Cells that are negative for both Annexin V and the vital dye have no
indications of apoptosis: PS translocation has not occurred and the plasma
membrane is still intact.
Early Apoptosis
Cells that are Annexin V-positive and vital dye-negative, however, are in
early apoptosis as PS translocation has occurred, yet the plasma
membrane is still intact.
• Late Apoptosis or Cell Death
Cells that are positive for both Annexin V and the vital dye are either in the
late stages of apoptosis or are already dead, as PS translocation has
occurred and the loss of plasma membrane integrity is observed.
When measured over time, Annexin V and a vital dye can be used to
monitor the progression of apoptosis: from cell viability, to early-stage
apoptosis, and finally to late-stage apoptosis and cell death.
13.
14. LDH ASSAY
• LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of
lactate to pyruvate. In the second step of the reaction, diaphorase uses the
newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt
(INT) to highly-colored formazan which absorbs strongly at 490-520 nm.
Test principle
The assay is based on consideration that tumor cells possess high concentration of
intracellular LDH and the cleavage of a tetrazolium salt when LDH is present in the
culture supernatant.
15. Quantitative value for the loss of cell viability
The activity of LDH can be measured as the reduction of
pyruvate to lactate.
The reduction is coupled to the oxidation of NADH to NAD+,
which is followed spectrophotometrically at 340nm
LDH
Pyruvate + NADH + H+ ⇌ NAD+ + lactate
As NADH has a high absorbance at 340nm compared to NAD+,
the reaction is measured as the rate of decrease in absorbance at
340nm.
LDH (lactate dehydrogenase) Leakage
17. MTT Assay
Introduction
This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliferation
and activation of cells.
The assay is based on the capacity of mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a
dark blue formazan product which is insoluble in
water.
18. The amount of formazan produced is directly
proportional to number of viable cells present in the
sample.
metabolically active Cell
MTT Formazan
Insoluble
19. Materials and equipment
MTT solution (5 ㎎/㎖ in phosphate buffered saline (PBS) pH 7.5),
HCl, Propan-2-ol
96-well microtiter plate, ELISA reader
Procedure (suspension and monolayer cells)
1. Prepare MTT stock solution and filter through a 0.2 ㎛ filter to
sterilize and remove the small amount of insoluble residue
2. To 100 ㎕ cell suspension or cell monolayer in each microtiter well
add 10 ㎕ MTT
3. Incubate in a humidified incubator at 37℃ for 3 h
4. Add 100 ㎕ 0.04 M HCl in propan-2-ol to each well and mix
thoroughly to dissolve insoluble dark blue formazan crystals
5. Read plate on a ELISA reader using a test wavelength of 570 nm
and reference wavelength of 630 nm
20. Compare with MTT assay and XTT assay
Culture cells in a MTP
for a certain period of time (37℃)
MTT assay XTT assay
Prepare labeling mixture
Incubate cells (0.5-4 h, 37℃)
Add solubilizing solution
(Isopropanol) and incubate
Measure absorbance using an ELISA reader
Add XTT labeling mixtureAdd MTT labeling reagent
Insoluble formazan Soluble formazan
21. Example: MTT and XTT
MTT XTT
Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002.
Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for
quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73
22. Principle
Dye elution:
Cell up-taken dye was measured colorimetric method after acetic
acid dye elution.
Nuclei counting
Incubation of cell samples in a mixture of citric acid and crystal
violet causes cells to lyse and the released nuclei to stain purple.
Crystal violet
23. Procedure
Dye elution
① After removal of medium, rinse
96 well plates with 100 ㎕/well
of PBS and stain with 100 ㎕
0.25% (g/10ml) aqueous crystal
violet for 10 min.
② Rinse plates four times in tap
water.
③ Dry the outsides of the plates
with paper to help avoid water
stains, and then dry the plates at
37℃. When dry, add 100 ㎕ per
well of 33% glacial acetic acid
(33 ml/100ml) and mix the
contents of each well before
reading at 570 nm.
Nuclei counting
① Collect the cells from animal
and centrifuge it.
② Remove clear supernatant by
aspiration.
③ Add 1ml of crystal violet
reagent.
④ Incubate at 37℃ at least 1 h.
⑤ Introduce a sample into the
hemocytometer chamber and
count the purple-stained nuclei
as for whole cells.
24. Acid phosphatase (AP) assay
The action of this enzyme in many of tissue is to cleave a
waste product called pyrophosphate and effectively
convert it to a useable phosphate.
P-nitrophenyl phosphate will be the substrate and
nitrophenol is the product of this reaction.
Nitrophenol is colorless but when the pH of the reaction
solution is alkaline, it is appears yellow. The pH of the
reaction solution will be changed by the addition of NaOH.
26. Materials and equipment
Substrate-containing buffer : 10 mM P-nitrophenyl phosphate in
0.1 M sodium acetate pH 5.5, 1 M NaOH
96-well micro titer plate, Microplate reader
Procedure
1. At end of cell growth period, remove medium and rinse wells in
100 ㎕ PBS
2. Add 100 ㎕ substrate-containing buffer to each well
3. Incubate for 2 h in incubator. Read plates at 405 nm, and either
reincubate for a further time if increased sensitivity is required, or
‘stop’ with addition of 50 ㎕/well of 1 M NaOH to cause an
electrophilic shift in the p-nitrophenol chromophore and thus
develop the yellow color, giving greatly increased sensitivity
27. Principle
In the presence of cellular metabolism the color of Alamar Blue (ALB) changes
from a fully oxidized, non fluorescent blue to a fully reduced, fluorescent red.
ALB will be reduced by a variety of enzymes and small molecules, including
the cytochrome system, FMN, FAD, NAD, and NADP.
Advantages
Simple, rapid, inexpensive, required no lysis, extraction or washing of sample
Disadvantages
Unstable during storage
Characteristics
- Sensitivity :
- The ALB assay is faster, simpler, and less artifact prone than the MTT assay.
Alamar Blue oxidation-reduction assay
28. Procedure
① At the end of an experimental incubation period, add 1 vol of ALB stock solution
per 25 vols (4%v/v) of growth medium in each well (8 ㎕ ALB for 200 ㎕ of
growth medium)
② Incubate plates at 37℃ for 3 h to allow metabolic dye reduction.
③ Equilibrate plates to room temperature for 30 min in the dark.
④ Measure the relative fluorescence at 530~560 nm excitation and 590 nm emission
wavelengths. Fluorescence is temperature sensitive; either equilibrate plates in a
warm room at the culture incubation temperature.. The ratio of test to control
fluorescence values at 590nm measures the effect of a treatment on cell growth or
metabolism.
⑤ For spectrophotometric assays, correct for the spectral overlap of the oxidized
and reduced forms of ALB by measuring each sample at two different
wavelengths, between, approximately, 540~630 nm . One of these must be a low
wavelength (LW) and the other a high wavelength (HW); for example, 570 ~ 600
nm, respectively.
29. ⑥ A correction factor (RO) for the absorbance of oxidized ALB must be
calculated.
ⓐ Measure the absorbance (AM) of growth medium alone. (no ALB)
ⓑ Measure the absorbances of oxidized (blue) ALB in growth medium at the low and
high wavelengths.
ⓒ Substract AM from each of the measured ALB absorbance to produce, respectively,
AOLW and AOHW , the absorbance of oxidized (blue) ALB at the low and high
wavelengths.
ⓓ Calculate the correction factor RO of oxidized ALB:
RO=AOLW/AOHW
⑦ Measure the absorbance values (ALW and AHW) of a test sample at each
wavelength.
⑧ Calculate the percentage of reduced ALB (ARLW) in a sample as:
ARLW = 100 x [ALW-(AHW x RO)].
⑨ Calculate the percentage difference in reduction (PDR) between treated
and control cells: PDR = 100 x (test ARLW/ARLW for positive growth control)
30. Neutral Red assay
(3-amino-7dimethyl-2-methyphenazine hydrochloride)
Principle
- The incorporaton of NR into the lysosomes of viable
cells after their incubation with test agents.
Use
- Industrial, pharmaceutical, environmental and other
testing laboratories concerned with acute toxicity
testing.
Advantages
- Simplicity, speed, economy, and sensitivity
31. Materials and Equipments
Solution
① Neutral red
4mg/ml stock solution
Dilute 1:100 into medium , incubate
overnight at 37℃ and centrifuge for
10 min at 1500 g before use.
② 1% CaCl2/0.5% formaldehyde
Mix 6.5 ml 37% formaldehyde with
50 ml 10% CaCl2 and 445 ml distilled
water.
③ 1% acetic aicd/50% ethanol
Mix 4.75 ml acetic acid with 250 ml
95% ethanol and 245 ml distilled
water.
Equipment
① Complete media suitable for
chosen cell type.
② Culture petri dish
③ 96well tissue culture plate
④ Inverted microscope
⑤ ELISA-type
spectrophotometer
⑥ Microplate shaker
⑦ Eight-channel pipette
32. Procedure
① Resuspend cells of actively growing culture and count cells and accurately
allocate appropriate number suspended in medium.
② Seed 0.2 ml containing desired number of cells to each well of 96 well plate and
incubate at 37℃ for 24 h or longer.
③ Remove the medium and add fresh medium containing graded dilutions of test
agent. Incubate for desired length of time. Examine at least 4-8 wells per
concentration of test agent.
Keep serum concentration as low as possible during this step.
④ After incubation for desired time interval, remove medium with test agent and
incubate cells with fresh medium containing 40 ㎍/ml NR dye.
⑤ Continue incubation for 3h to allow for incorporation of vital dye into survival
cells.
⑥ Remove medium by inverting the plate and rapid rinse with a mixture of 1%
CaCl2 / 0.5%formaldehyde.
⑦ Extract dye into supernatant with 0.2 ml of solution of 1% acetic acid/50%
ethanol.
After10 min at room temperature and rapid agitation for a few seconds on a
micrometer plate shaker, scan the plate with an ELISA-type spectrophotometer
equipped 540 nm filter.
34. Principle
The rate of DNA synthesis is a reflection of
proliferation under many condition.To measure the
proliferative rates by [3H]-thymidine uptake, cells
are cultured in microtitre wells, thymidine is
added, and the uptake by DNA is measured , after
lysing and washing on, by scintillation counting.
Bromodeoxyuridine(BrdU) can be incorporated
instead of [3H]-thymidine and the incorporation
can be assayed with antibodies to BrdU in a non-
radioactive assay.
[3H]-thymidine and BrdU incorporation
(DNA synthesis measurement)
36. Labeling index with [3H]-thymidine
① Set up the culture at 2x104 cells/ml~ 5x104 cells/ml in 24 well plates
containing cover-slips. Grow to the desired cell density.
② Add [3H]-thymidine to the medium and incubate the cultures for 30
min.
③ Remove the labeled medium, and discard it into a designed container
for radioactive waste.
④ Wash the cover-slips three times with PBSA.
⑤ Add 1:1 PBSA: acetic methanol, 1ml per well, and remove it
immediately
⑥ Add 1ml of acetic methanol to each well, and leave the cultures for
10min.
⑦ Remove the cover-slips, and dry them with a fan
⑧ Mount the cover-slip on a microscope slide with the cells uppermost.
⑨ Leave the mountant to dry overnight.
37. 37
Terminal Deoxyribonucleotidyl Transferase-
Mediated dUTP Nick End Labeling (TUNEL) assay
* Terminal deoxyribonucleotidyl transferase (TdT) can
catalyze the addition of nucleotide at 3’ OH end of DNA.
* Cells are incubated with TdT, Co2+,
biotinylatedmdeoxyuridine triphosphate.
* Cells are then incubated with fluorescently labeled
streptavidin.
*Apoptotic cells can be detected by fluorescence microscopy
or flowcytometry.
38. Incorporation of fluorescein-dUTP to
3’-OH DNA ends using
enzyme Terminal deoxynucleotidyl Transferase (TdT)
5’ 3’OH
dUTP****
TUNEL ASSAY
TdT-mediated dUTP Nick-End Labeling
Detection of DNA Degradation
39. dNTP dUTP
Direct Indirect
X Fluoresein, FITC, PE etc Biotin DIG
Avidin conjugated with
fluoresein, AP, POD
Anti-DIG antibody
conjugated with fluoresein,
AP, POD
Ⅰ. Enzymatic DNA labeling
40. Comet Assay
The single cell gel electrophoresis (SCGE), the
Comet Assay, is a fairly simple procedure by using a
micro gel and electrophoresis to detect DNA damage.
After image analysis the damage in DNA looks like a
comet, so that’s why it is known as Comet Assay. This
technique is further developed and introduced the use of
high alkaline conditions. This step increased the ability
of the assay to detect not only the double strand breaks
but also the single strand breaks.
To detect genotoxicity of the drug
44. • Tail length:
It is defined as a measurement from the point of
greatest intensity within the comet head.
• Tail moment:
It is defined as the product of the tail length and
the fraction of total DNA present within the tail.
TM = tail length*100
head
• Tail intensity:
It is defined as the florescence detected by image
analysis in the tail, which is proportional to the amount
of DNA that has moved from the head region into the
comet tail.
46. PARP CLEAVAGE ASSAY: WESTERN BLOT AND FLOW CYTOMETRY
Caspase-3 cleaves many cellular proteins including PARP (Poly (ADPRibose)
Polymerase). PARP is a 116 kDa nuclear protein which is strongly activated
by DNA strand breaks
.
PARP plays a role in DNA repair as well as in other cellular processes,
ncluding DNA replication, cell proliferation and differentiation.
During apoptosis,caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25
kDa fragment. PARP cleavage is considered to be one of the classical
characteristics of apoptosis.
An anti-PARP-FITC conjugated Cleavage Site-Specific Antibody (CSSA) (Cat.
#44-699) that can detect apoptotic cells by flowcytometry. An alternative to
the TUNEL assay, the PARP-FITC CSSA can detect apoptosis in adherent and
suspension cells.
47. PARP CLEAVAGE ASSAY: WESTERN BLOT AND FLOW CYTOMETRY
Caspase-3 cleaves many cellular proteins including PARP (Poly (ADPRibose)
Polymerase). PARP is a 116 kDa nuclear protein which is strongly activated
by DNA strand breaks
.
PARP plays a role in DNA repair as well as in other cellular processes,
including DNA replication, cell proliferation and differentiation.
During apoptosis,caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25
kDa fragment. PARP cleavage is considered to be one of the classical
characteristics of apoptosis.
An anti-PARP-FITC conjugated Cleavage Site-Specific Antibody (CSSA) that
can detect apoptotic cells by flowcytometry. An alternative to the TUNEL
assay,
PARP Universal Colorimetric Assay measures the activity of PARP in cells
and tissues by detecting the incorporation of biotinylated Poly (ADP-ribose)
onto histone proteins.
48. ENDONUCLEASE ASSAY
Principle;
During apoptosis the endonuclease enxymes are activated to
Cleave the genomic DNA in to many smaller fragments
Steps
1. Preparation of agarose gel containing
Ethidium bromide and genomic DNA
2. Making well
3. Preparation of cell lysate
4. Incubation in humidified atmosphere
after loading cell lysate
5. Observe the DNA degradation
49. Detection of Caspase-3: Fluorometric Assay
Activation of proteases/caspases initiates apoptosis in mammalian cells.
The Caspase-3 Fluorometric Protease Assay provides a simple and
convenient means for assaying the DEVD-dependent caspase activity.
The assay is based on detection of cleavage of substrate DEVD-AFC (AFC:
7-amino-4-trifluoromethyl coumarin). DEVD-AFC emits blue light ( max =
400 nm); upon cleavage of the substrate by related caspases, free AFC
emits a yellow-green fluorescence ( max = 505 nm), which can be
quantified using a fluorometer or a fluorecence microtiter plate reader.
Comparison of the fluorescence of AFC from an apoptotic sample with an
un induced control allows determination of the fold increase in caspase-3
activity.
BioVision
50. COLORIMETRIC METHOD: CASPASE-3 DETECTION
The substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pNA), and a
synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid
sequence of the Caspase-3 cleavage site in PARP. Upon cleavage of the substrate by
Caspase-3 or related caspases, free pNA light absorbance can be quantified using a
spectrophotometer or a microplate reader at 400 or 405 nm.
Comparison of the absorbance of pNA from apoptotic sample with an uninduced control
allows determination of the increase in Caspase-3 activity.
Bio Source
53. Fig. In Hoechst 33258 / PI double staining, cells with blue intact
nuclei were viable cells, whereas those with blue fragmented nuclei
were early apoptotic cells. Cells with pink intact nuclei were necrotic
cells, whereas cells with pink fragmented nuclei were late apoptotic
cells. (blue against Hoechst33258, red against PI)
Apoptotic(0%)
Necrotic(10.5%)
Apoptotic(85.2%)
Necrotic(11.2%)
Apoptotic(1.2%)
Necrotic(92.5%)
56. Clonogenic Cell:
Defined as a cell with the capacity for sustained
proliferation
Have undergone a minimum of 5-6 doublings to give
rise to colonies containing at least 50 cells
Colony-forming Efficiency
57. Colony-forming Efficiency (CFE)
The ability to form colonies is used as a measure of
reproductive integrity
It is often referred to as plating efficiency (PE)
Number of colonies formed
CFE = 100%
Number of cell plates
Clonogenic assays
The basis of assays for determining the lethal effects of
cytotoxic agents
58. Determining the PE of an established
adherent cell line
Materials and Equipment
Cell growth medium : Eagle’s basal medium (BME)
100 iu/ml penicillin,
0.1 mg/ml streptomycin
Trypsin-EDTA
Gentain violet stain
59. Procedure
1. Trypsinize monolayer cultures or use cell suspension
cultures and determine the viable cell count
2. Dilute cells in growth medium to 1000 , 2000 and
5000 cells/10ml
3. Inoculate nine replicate Petri dished with 4 ml growth medium plus
1ml cell suspension
4. Place plates in a humidified 5% CO2 plus air incubator are normal
growth temperature and rock shelf or tray gently to and fro three
times. The plates must not be moved now until colonies are stained
5. Stain and count three replicate per cell density at 1,2 and 3 weeks
(murine lines) or 2 , 3 and 4 weeks (human lined)
6. Calculate the optimum cell densities for seeding and duration of
incubation
60. Example; Rat keratinocytes
(A) (B)
(C) (D)
Colony forming Non-colony
forming
48 hr after
subculture
6 days after
subculture
: colony , : Single cells