Technique / Cell Biology / Apoptosis assay

Apoptosis is programmed cell death found in multicellular organisms. It is one of the main types of programmed cell death and involves a series of biochemical events leading to a characteristic cell morphology and death.

Apoptosis protocols
(Curr Protoc Cell Biol.)
1: Curr Protoc Cell Biol. 2004 Feb;Chapter 18:Unit 18.8.

Flow cytometry of apoptosis.

Pozarowski P, Grabarek J, Darzynkiewicz Z.

School of Medicine, Lublin, Poland.

Common methods applicable to flow cytometry make it possible to: (1) identify and
quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or
necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell
morphology and chromatin condensation, which occur during apoptosis, can be
detected by analysis with laser light beam scattering. Early events of apoptosis,
dissipation of the mitochondrial transmembrane potential and caspase activation,
can be detected using either fluorochrome reporter groups or appropriate
antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma
membrane can be detected by the binding of fluoresceinated annexin V. Another
apoptotic event, DNA fragmentation based on DNA content of cells with fractional
("sub-G1") or DNA strand-break labeling, TUNEL; or In Situ End Labeling, ISEL;.
Still another hallmark of apoptosis is the activation of tissue transglutaminase
(TGase), the enzyme that crosslinks protein and thereby makes them less
immunogenic. The major advantage of flow cytometry in these applications is that
it provides the possibility of multiparametric measurements of cell attributes.

2: Curr Protoc Cell Biol. 2001 Nov;Chapter 18:Unit 18.3.

Assessment of apoptosis and necrosis by DNA fragmentation and morphological
criteria.

Zhivotosky B, Orrenius S.

Karolinska Institute, Stockholm, Sweden.

Apoptotic cells share a number of common features, such as phosphatidylserine
(PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and
formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit
nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown
of cytoplasmic structure and organelle function, and cytolysis by swelling. This
unit describes some of the techniques most commonly used to detect cell death. A
number of assays are used for characterizing and distinguishing apoptosis and
necrosis. Morphological assays include trypan blue exclusion, differential
staining, and Hoechst staining. Methods to detect chromatin cleavage include
TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays
using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by
agarose gel electrophoresis, phenol extraction of DNA for analysis of
fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA
fragmentation by pulsed-field gel electrophoresis. A protocol is also provided
for Cytospin preparations from cell suspensions.

3: Curr Protoc Cell Biol. 2001 Aug;Chapter 18:Unit 18.2.

Analysis of caspase activation during apoptosis.

Kaufmann SH, Kottke TJ, Martins LM, Henzing AJ, Earnshaw WC.

Mayo Clinic, Rochester, Minnesota, USA.

This unit describes three methods for the detection of caspase activation as
cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are
provided using suitable substrates. Because the various low-molecular-weight
substrates available for these assays are not selective, however, the assays do
not accurately distinguish between various caspases. Immunoblotting is described
for following the activation of specific caspases. When coupled with subcellular
fractionation, this method can provide large amounts of temporal and spatial
information about caspase activation. Finally, affinity labeling protocols are
provided for detecting active caspases in whole-cell lysates or subcellular
fractions.

4: Curr Protoc Cell Biol. 2001 Aug;Chapter 18:Unit 18.1.

Current concepts in cell death.

Zhivotovsky B, Orrenius S.

Karolinska Institutet, Stockholm, Sweden.

This unit attempts to define cell toxicity that leads to cellular demise, with a
strong emphasis on cell death via both apoptosis and necrosis, by summarizing
some of the more recent developments in cellular, molecular, and biochemical
studies of the events that govern the induction and execution of cell death.
Specific topics pertaining to cell death include structural changes,
macromolecular degradation, cellular signaling, the role of mitochondria, and
genetic modulation of apoptotic cell death.

Cell cycle books
(Biowww Bookshelf)
Cell cycle related research

Apoptosis and cell proliferation books
(Biowww bookshelf)
apoptosis and proliferation research books

Figure. Fragmented DNA can be labeled by terminal deoxynucleotidyl transferase (TdT) for apoptosis
(Immunobiology)
Appendix I. Immunologists' Toolbox Characterization of lymphocyte specificity, frequency, and function.

Figure. The caspase cascade involved in apoptosis
(Molecular Biology of the Cell)
IV. Internal Organization of the Cell 17. The Cell Cycle and Programmed Cell Death Programmed Cell Death (Apoptosis)

Programmed Cell Death (Apoptosis)
(Molecular Biology of the Cell)
IV. Internal Organization of the Cell 17. The Cell Cycle and Programmed Cell Death

Death Domain Proteins
(Cancer Medicine)
Part II Scientific Foundation, Section 1: Cancer Biology 4. Apoptosis and Cancer

BrdU/TUNEL double staining
(Forum)
endothelial cells double staining for BrdU and TUNEL