Introduction to apoptosis assays in cell lines:
The word apoptosis is a Cellular suicide or
programmed cell death, by biochemical events which leads to cell changes in
morphology causing cell death. Apoptosis is morphologically characterized by
cytoplasmic shrinkage, nuclear compaction of chromatin, formation of membrane
bounded apoptotic bodies, and engulfment of cell by phagosomes.
Apoptosis
assays involves the “assessment
of apoptosis”. The standard method of identification and quantification is the
morphological assessment and biochemically, apoptosis is characterized by formation
of fragments in genome and degradation and cleavage in many cellular proteins.
The cell lines are the cell cultures which
are permanently established and proliferate indefinitely by giving appropriate
space and fresh medium. These cell lines are important to study
pathophysiological, physiological and differentiation process of specific cells.
It will allow us to examine the stepwise alteration of biology, structure and
genetic makeup of the cell under controlled environment. Cell lines also
undergoes apoptosis and in this subject we will discuss about apoptosis assays
in cell lines, so before going towards apoptosis analysis, apoptosis mechanisms
should take into consideration.
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General Apoptotic pathway
Mechanisms of apoptosis:
The proteolysis of almost 400 proteins by caspases (cysteine-aspartate
proteases) initiates cell death during apoptosis. Extrinsic and intrinsic cell
death pathways activate the caspases.
1 Intrinsic cell death pathway:
The intrinsic cell death pathway is also known as ‘‘mitochondrial
dependent cell death’’. This pathway is stimulated by stimulus like stress or damage
which may lead the cell to apoptosis which include damage in its DNA, oxygen
deprivation and other stresses which impaired cell’s ability to function.
During these response to stress and damage cell decides that its continuous
existence may be dangerous to other organisms as a whole so, it activates a set
of proteins ‘Bcl-2 protein family’ which is involved in regulated apoptotic
cell death consisting of anti-apoptotic and pro-apoptotic members. The
anti-apoptotic complex ‘Bcl-2 and B-XL’ prevent apoptosis by sequestering of
cell death driving cysteine proteases called caspases, or by preventing the
release of mitochondrial apoptotic factor such as “cytochrome c and AIF(
apoptosis inducing factors) into the cytoplasm. By entering the cytoplasm,
cytochrome c and AIF directly activate caspases which cleave set of cellular
proteins and cause apoptosis.
In contrast, the pro-apoptotic members of this
family “Bax and Bak” initiate the release of caspases which in turn cause the
increase in permeability of mitochondrial membrane and cause cell death. So,
the Bcl-2 protein family acts as “life-death” decision point in the common
apoptotic pathway. The key step involves the intrinsic
cell death pathway is actually the permeabilization of the outer mitochondrial membrane,
after which the cells are committed to death.
The
caspase 8 activated during extrinsic cell death path make the bid protein into
activated bid protein which involves in the mitochondrial cell death pathway by
releasing the cytochrome c and AIF from inner mitochondrial membrane into
cytosol. The cytochrome c interact with Apaf-1 and procaspase 9 and make it an
activated apoptosome. This apoptosomes in turn convert the procaspase 3 to
activated caspase 3, which transform the apoptotic factor into activated
apoptotic factor which initiates the apoptosis in the cell through intrinsic
pathway.
1 Extrinsic cell death pathway:
Activation
of extrinsic cell death pathway occurs by following the binding of
ligand fas and tumer necrosis factors-a (TNF)
on the cell surface of “death receptors” .These death receptors recruit the adaptor
molecules such as FADD and procaspase 8 which is activated into caspase which
then activate caspase 3 and caspase 7, leading to apoptosis.
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Intrinsic and extrinsic apoptotic pathway of cell
death
Methods and
techniques used in apoptosis assay:
- Loss of membrane asymmetry.
- Apoptosis related proteases: caspases.
- Mitochondrial
transmembrane potential (ΔΨm).
- Cytochrome c
release.
- Nuclear
condensation, DNA fragmentation and increase of subG1 population.
- Nuclear cell
membrane blebbing.
Parameters of apoptosis
1.
Loss of membrane asymmetry:
Loss
of membrane symmetry is the early sign of apoptosis which involves the
embedding of phosphatidylserine(PS) in membrane which is now externalize and signal
the phagocytosis.
Annexin V apoptotic assay:
Annexin
V is the placental protein in humans which binds with the PS in the presence of
calcium. The flourochrome-conjugated annexin V is the most commonly used tool
to detect and quantify the PS exposure and indicate membrane asymmetry.
Normally the binding of annexin V is paired with the cell viability reagent
propidium iodide (PI) which is normally not able to penetrate plasma membrane
and can differentiate between apoptotic and necrotic cells.
Flow
cytometry or fluorescence microscopy techniques can be used to detect the
binding of flourochrome-conjugated annexin V with PS. The quick and accurate
quantification of cells could be done most successfully by flow cytometry and
flourecence microscopy will allow the visualization of apoptotic events in cell
lines.
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Membrane
asymmetry detection by annexin V assay
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The
caspase family is a group of cysteine proteases which plays a role in
apoptosis. The mammalian caspases are subdivided into three subgroups:
- Initiator caspases (2, 8, 9 and 10) which initiate the apoptosis signals.
- Executioner caspases (3, 6 and 7) which carry out the mass proteolysis and moves towards the apoptosis.
- Inflammatory caspases(1, 4, 5, 11 and 12) not involves in apoptosis but in inflammatory cytokines signaling.
Initially
synthesized pro-caspases become activated after cleavage and show response to
granzyme B, death receptor, and apoptosome stimuli. Caspases then cleave many
substrate including nuclear protein, downstream caspases, plasma membrane
proteins, mitochondria proteins and ultimately leading to cell death.
Caspase activation detection:
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Sample
type
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Detection
method
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Best
to use when you want to
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Live cells (adherent or suspention)
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Flow cytometry
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Quickly quantify and detect how many
cells possess active caspase using specific substrate.
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Fixed cells(adherent or suspension)
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Fluorescence microscopy
Flow cytometry
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Detect cells having active caspase and
visualize the other proteins at the same time.
Quickly quantify and detect how many
cells possess active caspase using specific antibody.
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Cells or tissue lysate( fresh or
frozen)
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Western Blot
Absorbance/ Fluorescence Assay
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Detect caspase in cleave form and
pro-caspase using specific antibody.
Quickly detect caspase activation in
cell population using specific substrate.
Detect caspase activation using
specific antibody against the active form.
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Tissue sections (frozen or paraffin)
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IHC
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Detect caspase activation using
specific antibody in descript cell in heterogeneous tissue.
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Caspase activation detection
3.
Mitochondrial trans-membrane
potential (ΔΨm):
One
of the distinctive features to characterize apoptosis is the disruption of
mitochondrial membrane function, specially the change in membrane potential.
Membrane potential is critical for maintaining the normal physiological
functioning of the respiratory chain and production of ATP. Opening the
mitochondrial permeability transition pore (MPTP) leads to change in membrane
potential and cause the release of the cytochrome c.
Mitochondrial
membrane potential is detected by cationic (positively charged) fluorescent dye
that is accumulated in negatively charged mitochondrial matrix. The amount of
dye accumulation is inverse proportion to the membrane potential: means more
negative membrane potential more dye will accumulate, so we say that the
healthy cell contain more dye than then apoptotic cells.
These
dyes are used qualitatively in fluorescence microscopy and quantitatively in
flow cytometry or micro plate spectrophotometry. The mitochondrial membrane
potential probes are also available.
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Probe
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Healthy/apoptotic
cell
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Best
to use for
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TMRE(Tetramethylrodamine,
ethyl ester) cationic red orange dye which readily accumulate in healthy mitochondria,
depolarized mitochondria have less membrane potential an unable to sequester
TMRE.
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Healthy cell: bright orange
fluorescence.
Apoptotic cell: weak orange fluorescence.
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Immunofluorescence staining and
time-lapse fluorescence microscopy.
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JC-1:
Cationic green-red dye
which determine potential dependent
Accumulation in mitochondria. Decrease
in mitochondrial depolarization is indicated by decreased red/green
fluorescent intensity ratio.
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Healthy cell: red
Apoptotic cell: green
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Micro plate spectrophotometry or Flow
cytometry . Ideal for comparative measurements.
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JC-10:
a derivative of
JC-1, have increased solubility in
aqueous media and have the ability to detect subtler changes in mitochondrial
membrane potential loss.
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Healthy cell; orange fluorescence.
Apoptotic cell: green.
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Flow cytometry or microplate
spectrophotometry. Ideal for comparative measurements.
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MitoOrange
dye and Mito NIR dye: cationic dye, in normal cell
fluorescence intensity increase when dye accumulate in normal cell, while the
intensity decrease in apoptotic cell following collapse in membrane
potential.
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Healthy cells: red or near infrared(NIR) fluorescence.
Apoptotic cell:
weak fluorescence.
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Microplate spectrophotometry or flow
cytometry.
Multiparametric study of apoptosis.
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Change in
trans-membrane potential causing cytochrome release
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4.
Cytochrome c release:
The
change in membrane potential of mitochondria is fairly a catastrophic event
which leads to the opening of mitochondrial permeability transition pore in
membrane and cause the subsequent release of the cytochrome c into cytosol
which triggers the downstream event in apoptotic cascades. When the cytochrome
once released into cytosol the cell is reached to “point of no return” from
where it is impossible for cell to recover from apoptosis and death is most
likely to occur.
The
most common technique which is used to detect the release of cytochrome c is
through western blot on protein which is extracted from different sub cellular
compartments. It is important to make sure that the subcellular fractions we
are going through are not contaminated by other fractions. The reliable sub cellular
markers are:
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Cytoplasmic markers: GAPDH,
actin
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Mitochondrial markers: VDAC1, PDH-E1
5. Nuclear condensation, DNA
fragmentation and increase of subG1
population:
Chromatin
condensation:
Chromatin
condensation, DNA fragmentation, with cell membrane blebbing is the
morphological indicator of terminal stages of apoptosis. During the process of
apoptosis the chromatin material undergoes from a heterogeneous genetically
active form to highly condense inert form. After staining with DNA-binding
nuclear dye, the compacted DNA will be stained brightly than the active
non-apoptotic cell having active genome. The detection of condensed nuclei can
be done qualitatively through fluorescence microscopy and quantitatively through
flow cytometry.
Genomic DNA
fragmentation detection through Agarose gel electrophoresis and TUNEL assay:
The
condensed DNA can be fragmented by specific nucleases Caspase Activated DNAases
(CAD). These enzymes activated through caspase cascade leads to cleavage of DNA
at internucleosomal linker sites in the nucleosomes and generate fragments of
~200 base pair known as ladder.The classical method for detection of the genome
fragments is on the agarose gel. Agarose gel electrophoresis is an semi
quantitative method that provide us a robust answer.
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| DNA fragmentation detection through TUNEL assay |
The
most commonly used assay for fluorescent microscopy and flow cytometry is the
TUNEL assay is an alternative method to detect the DNA fragmentation which
involves the identification of nicks by this assay. The TdT DUTP nick and
labeling technique is mainly based upon the ability of terminal
deoxynucleotidyl transfersae (TdT) to end labeling of nicks in DNA brought
about by the caspase activated deopxyribonuleases. TdT is the enzyme which have
the ability for labeling 3’ blunt end of double stranded DNA with deoxyuridine.
Increase of subG1 population:
The increase in the
number of the cells which are stalled at G1 is also the consequence of the DNA
fragmentation, and is readily detected with flow cytometer. When we
permeabilize the cells with 70% ethanol the DNA will leak out leaving the cell
population with reduced DNA content. When we stain the cells with DNA staining
dye like PI, the DNA profiling by flow
cytometry
show the DNA at different cell stages (G1, S, G2 and M).We can easily identify
the apoptotic cells as the subG1 population seen to the left of the G1 peak. So the G2 includes the entire
dead cell in the population irrespective of the type of the cell. So, this Para
meter also helpful in apoptotic detection.
6. Nuclear cell membrane blebbing:
Cell
blebbing and contraction along with fragmentation is the best indicator of the
apoptosis in cell. It occurs mainly because of the cytoskeleton breakage
causing the membrane bulge outward. This bulge eventually separated from the
cell taking the part of the cytoplasm and is now known as the apoptotic bodies.
Phase-contrast microscopy can be used to detect the membrane blebbing. However
this is an indirect method and may give you false positive or negative
response.
Why we use
apoptotic assays?
Apoptosis
or programmed cell death is a normal or natural process in normal cells to
maintain homeostasis in the cell lines, organs and tissue, in vitro or inside
the body. Apoptotic assay is actually the analysis, assessment, test or checks
to determine the programmed cell death by various signaling pathway, so we use
different analysis techniques and method to understand and evaluate the process
of apoptosis. Different type of assay are used to determine the apoptotic cell
at different level of apoptosis and helpful to understand the normal cell
processes undergoing inside the cells, and any deviation from the normal
processes during cell lines.
The pros and cons of apoptosis assays:
The apoptosis assays are used widely nowadays to
examine the cells deviating from normal mechanics of development and going
towards programmed cell death.We use many type of assays to determine the
different morphological and biochemical changes in the normal cell during
apoptosis. These assays are useful in many ways and may be some time taken
wrong like only carrying out a single assay to determine the apoptosis is not
enough, because it may misguide the observer so we should take the more than
one assay at a time to determine the apoptosis and meat the level of accuracy
and avoid the confusion between apoptosis and necrosis.And most importantly the
selection of the best assay is possible only by knowing the strengths and limitation
of the assay being used and it may eliminate all the limitations.
Summary:
In a nutshell, apoptosis is the natural
processes going out in the cells and tissues of living organisms and is mainly
related to homeostatic maintained inside the body when the cells are going
through the stress or diseased conditions the internal signaling pathway itself
make the cell to pass through the death, so we should have an idea that
homeostasis is not a passive process but is actively maintained through
apoptosis. During cell death pathway cell undergoes through many indications
like DNA fragmentation, membrane blabbing, mitochondrial potential change (ΔΨm),
and each of the changes which the cell undergo are detected by different
analysis techniques called apoptotic assays. The most useful assay is the TUNEL
assay used to detect the DNA fragmentation during apoptosis. So, these assyas
are helpful to check many cell cultures, cell lines and help us to
differentiate that the cell is going through the apoptosis not the necrosis.
Apoptotic assay may be advantageous or disadvantageous it depend upon the
selection of the assay like the assay used to detect the membrane blebbing may
not only be helpful because the blebbing in membrane may also be due to the
necrosis or other cause, so as far as our concern we should use more than one
assay to detection and positive results. Increased in the interest of the
apoptosis has resulted in the development of the new techniques and also the
revival of the existing, because it is most helpful in cell and tissue culture
labs as well.
References:
·
https://www.researchgate.net/deref/http%3A%2F%2Fwww.bioscience.co.uk%2Fuserfiles%2Fpdf%2FApoptosis_Necrosis_and_Cell_Viability_Assays.pdf
·
https://docs.abcam.com/ebooks/apoptosis-analysis-guide.pdf
https://www.rndsystems.com/resources/technical/tunel-assay-principle
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