This document summarizes and compares different methods for detecting apoptosis in cells, including morphological changes, membrane asymmetry, protease activity, and DNA modifications. For morphological changes, methods like confocal microscopy and phase contrast microscopy allow visualization of features like blebbing and apoptotic bodies. Electron microscopy also enables detection of nuclear density changes. Advantages are low cost and providing information for further studies, while disadvantages include lack of objectivity and difficulty quantifying apoptosis. Membrane asymmetry detection uses annexin V binding to phosphatidylserine exposed on the outer membrane of apoptotic cells. This can be analyzed via flow cytometry or fluorescence microscopy and distinguishes apoptotic from necrotic cells. Advantages are rapid accurate quantification

1. Comparison of apoptosis
detection methods
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2. CONTENTS
Morphological Changes
Membrane Asymmetry
Protease Activity
DNA Modifications

3. Part One
Morphological Changes01

4. 1-1 Methods
Another versatile tool is
confocal laser scanning
microscopy, powerful for both
morphological analysis and
macromolecular localization.
Membrane blebbing and
apoptotic bodies can also be
seen by simple phase-contrast
light microscopy.
The application of quantitative
digital imaging techniques to
electron microscopy enables
the detection of even subtle
changes in nuclear densities.
01
02
03

5. 1-2 Advantages
It is a relatively reliable and inexpensive method for detecting
apoptotic cells.
The plethora of information provided is wide, may be useful for
subsequent biochemical or molecular studies, furnishing important
controls for the experiment.

6. 1-3 Disadvantages
2
4
1 3
Quantitative measurement lacks objectivity
and reproducibility
Many samples cannot be analyzed, it
requires laborious preparation
The procedure is time consuming and is
fairly expensive
Because only a small area can be visualized,
quantification of the extent of apoptosis is
also difficult

7. Part Two
Membrane Asymmetry02

8. The binding of fluorochrome-conjugated Annexin V to exposed PS can be detected by flow cytometry or fluorescence microscopy.
Annexin V is a calcium-dependent protein that
preferentially binds phosphatidylserine, fluorochrome-
conjugated Annexin V is a commonly used tool to
detect and quantify the PS exposure characteristic.
Often, Annexin V binding is paired with cell viability
reagents such as propidium iodide (PI) that are
normally not able to penetrate the plasma membrane
to differentiate between apoptotic and necrotic cells.
2-1 Methods

9. 2-2 Advantages
• Easy, rapid and accurate quantitation of
apoptosis in both viable and fixed single
cells.
• Explain the relationship between
induction of apoptosis by different
agents and their cell cycle phase
specificity.
Membrane Asymmetry

10. Very time consuming as it has multiple steps and is
quantitative. Therefore, intact tissues usually require
pre-treatment with an enzyme to release the
individual cells for analysis.
Membrane Asymmetry
2-3 Disadvantages

11. Part Three
Protease Activity03

12. Characteristics
 Highly conserved cysteine
proteases.
 Subdivided into three functional
groups.
 Initially synthesized as inactive pro-
caspases.
3-1 Caspases

13. 3-2 Methods
The ability to detect active caspase relies on the specificity of the antibody and where
the epitope is located. If the antibody recognizes an epitope found in both the pro-
and active forms of the caspase, you will only be able to differentiate both forms by
visualization in Western Blot.
Antibody-based methods
Biochemical substrates contain a specific cleavage sequences that are recognized by the
caspase and are covalently attached to a colorimetric or fluorogenic detection probe. Upon
cleavage, the colorimetric or fluorogenic compound is liberated producing an increase in
absorbance (colorimetric substrate) or fluorescence light (fluorogenic substrate).
Substrate-based methods

14. Characteristics
 Calpains are cytosolic calcium-
dependent cysteine proteases
composed of one or two subunits.
 Cathepsins are generally found in
lysosomes, and most cathepsins
are cysteine proteases.
3-3 Calpain&Cathepsins
Calpain
Cathepsins

15. 3-4 Methods
Calpain and Cathepsin activity can be easily detected in many cell types using a specific
calpain/cathepsin substrate linked to a colorimetric or fluorogenic detection molecule
that will be released upon cleavage of the substrate.
Substrate-based methods

16. Protease Activity
Detection of active protease in situ is an easy, sensitive,
and reliable method for quantifying apoptosis.
Protease activation can be detected in a variety of ways
including western blot, immunoprecipitation and
immunohistochemistry.
This technique allows selection for individual initiator or
execution caspases. It also allows for rapid and consistent
quantification of apoptotic cells.
3-5 Advantages

17. 3-6 Disadvantages

18. Part Four
DNA Modifications04

19. 4-1 Methods
At the late stage of apoptosis, caspase-activated endonuclease cause
double-stranded DNA breaks. These apoptotic nucleosome fragments
can be resolved by gel electrophoresis as typical DNA ladders.
Gel Electrophoresis
TUNEL Assay
TUNEL assay utilizes TdT to mark those breakpoints with tagged
nucleotides, which are then detected using enzyme-tagged (for IHC)
or fluorescently labelled (for FACS) antibodies.

20. The lesions measured are
identifiable at the
molecular level.
Molecular Level
Precise in the determination
of cell death and DNA
damage.
Precise
DNA ladder assay and
TUNNEL staining have high
sensitivity.
Sensitivity
The assay can be applied to
study the very early events of
apoptosis.
Early Apoptosis
4-2 Advantages

21. Provide qualitative
rather than quantitative
results.
4-3 Disadvantages
The sensitivities and specificities
depend on fixative used, pre-
treatment and concentration of
terminal transferase enzyme.
These procedures have
multiple steps and
require more time.
Qualitative Results More Variate Time Consuming

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