Methods in Apoptosis
Apoptosis and necrosis are two mechanisms of cell
death, each with its own distinguishing morphological
and biochemical features. Necrosis, which occurs
within seconds of cell insult (Majno and Joris, 1995),
may be described as "cell murder" resulting from
external damage to the cell membrane, loss of homeostasis,
water and extracellular ion influx, intracellular
organelle swelling, cell rupture (lysis), and so inflammatory
cell attraction. Initially described by Kerr et al.
(1972), apoptosis is a much slower process of events
than necrosis, requiring from a few hours to several
days (depending on the initiator) and resulting from
molecular signals initiated within individual cells (see
Nagata, 1997; Barinaga, 1998; Van Cruchten and Van
Den Broeck, 2002). The initiators of apoptosis that
instigate the cascade of events leading to activation of
a series of cytoplasmic proteases, termed caspases
(cysteinyl-asparatate-specific proteinases), are multiple.
Two such pathways involve (i) activation of cell
surface death receptors, resulting in direct activation
of caspases, and (ii) cytochrome c release from the
mitochondria into the cytoplasm following induction
of leakiness in its membrane. The terminal caspases
downstream from these initiator mechanisms lead
to the morphological and biochemical events of
The mechanisms of apoptosis, which is analogous
to "cell suicide," are essentially the same, whether
induced by genetic signals or through external initiators.
The events involved include cell membrane blebbing;
chromatin aggregation; nuclear and cytoplasmic
condensation leading to cell shrinkage; and partitioning
of cytoplasm and nucleus into membrane-bound
apoptotic bodies, which contain ribosomes, morphologically intact mitochondria, and nuclear material. As
a result of the efficient mechanism for the removal of
apoptotic cells by phagocytic cells in vivo
, an inflammatory
response is not stimulated. In vitro
bodies swell and finally lyse (Darzynkiewicz and
Traganos, 1998; Kiechle and Zhang, 2002).
Apoptosis is key to many fundamental aspects
of biology, including embryonic development and
normal tissue homeostasis, as well as in many pathological
events, such as loss of regulated cell death in
cancer, response of cancer cells to chemo- and radiotherapy
(Clynes et al.
, 1998), and death of cells in diabetes
(Sesti, 2002) and neurodegenerative diseases
(Vila and Przedborski, 2003). Accurate detection of
apoptosis is of great importance to increase our understanding
of biological events that may allow us to
understand and to manipulate these events as a form
Major elements involved in the apoptotic pathway,
which should be considered when selecting
suitable methods for apoptosis detection, include the
Death receptor activation: Following receptor
cross-linking by ligand (e.g., the Fas receptor by the
CD95 (APO-1 Fas) or Tumor Necrosis Factor (TNF)
receptor type 1 by TNF), signal transduction leads to
caspase activation (see (f) below).
- Changes in cellular morphology: As described
- Membrane alterations: Translocation of phosphatidylserine
(PS) from the cytoplasmic to the extracellular
side of the cell membrane is an early event in
- DNA fragmentation: Prior to the induction of cell
membrane permeability, fragmentation of genomic
DNA at sites located between nucleosomal units, generating mono- and oligonucleosomal DNA fragments,
irreversibly commits the cell to die.
- Disruption of mitochondria: As described, disruption
of the mitochondrial membrane results in
cytochrome c (Apaf-2) release. This subsequently promotes
caspase activation by binding to Apaf-1 and
inducing activation of Apaf-3 (caspase-9). Similarly,
release of apoptosis-inducing factor (AIF) induces
- Activation of caspases: At least 11 different caspases
have been identified in mammalian cells. Activation
of this protease cascade via a range of stimuli is
central to the execution of apoptosis.
The range of techniques and methods for analysis
of apoptosis is extensive. Due to space limitations in
this article, we do not propose to describe all methods
comprehensively. Table I lists a number of techniques
for analysis of the cellular events described earlier. A
selection of techniques used for studying these events
is described in further detail.
II. MATERIALS AND
A. Light Microscopy (LM) and Fluorescence
Frost-ended slides and coverslips (Chance
Propper); ice-cold methanol; Coplin jars; forceps;
micropipettes; grease pen (DAKO S2002); and mounting
medium (Vectashield mounting solution with
antifade additive [Vector Labs.; H-1000]) suitable for
fluorescence slides and may also be used for LM slides.
Alternatively, 20% glycerol prepared in H2
O is also
suitable for mounting slides for LM.
If analysing suspension cells, a cytospin (e.g.,
Heraeus Labofuge 400) and cytospin cups are
For LM only
: haematoxylin, aluminium potassium
sulphate, citric acid, and chloral hydrate.
For FM only: Stains include 4',6-diamidino-2-
phenylindole (DAPI, Sigma D-9542), propidium iodide
(PI, Sigma P-4170), Hoechst 33258 (Sigma B-2883), Hoechst
33342 (Sigma B-2261), and acridine orange (AO, Sigma
A-6014) in phosphate-buffered saline (PBS), pH 7.4.
B. Gel Electrophoresis for DNA Ladder
Horizontal agarose gel electrophoresis chamber and
combs (Bio-Rad); electric power supply; UV transilluminator
or gel analyser (e.g., EpiChemi II Darkroom,
UVP Laboratory Products); PBS (Oxoid BR14a); ethidium bromide (Sigma E-8751); agarose (Sigma A-9539);
Tris (Sigma T-8524); EDTA (Tris E-5134); NaCl (Sigma
S-9899); sodium dodecylsulfate (SDS) (BDH 442152V);
RNase A (Sigma R-5250); proteinase K (Sigma P-2308);
boric acid (Sigma B-7901); bromphenol blue (Sigma B-
5525); glycerol (Sigma G-2025); molecular markers,
e.g., Phi X174 DNA Hae
III digest (Sigma D-0672);
C. Terminal Deoxynucleotidyl Transferase-
Mediated Deoxyuridine Triphosphate Nick
End-Labelling Assay (TUNEL)
Apoptosis detection system: (1) fluoresceincontaining
equilibrating buffer, (2) nucleotide mix, (3) TdT enzyme, (4) 20X SSC solution, (5) proteinase K, (6)
protocol (Promega; G3250); plastic coverslips, glass
slides and coverslips (Chance Propper); propidium
iodide (Sigma P-4170); Coplin jars; forceps; humidifying
chamber; 37°C incubator; Triton X-100 (Sigma T-
8787), PBS (Oxoid BR14a); 4% paraformaldehyde
(Sigma P-6148) in PBS (pH 7.4) (freshly prepared
Vectashield mounting solution with antifade
additive (Vector Labs.; H-1000); 70% ethanol [prepare
from absolute ethanol (Sigma E-7037)]; and
: Items 1-6 are included in the apoptosis detection
system available commercially from Promega
(G3250). There are, however, other detection kits available
commercially that may be equally suitable.
D. Reverse Transcriptase-Polymerase Chain
As all general laboratory glassware, spatulas, etc.,
are often contaminated by RNases, these items should
be treated by baking at 180~ for a minimum of 8h.
Sterile, disposable plasticware is essentially free from
RNases and so generally does not require pretreatment.
All solutions/buffers used should be prepared
in baked glassware using sterile ultrapure water
treated by the addition of diethylpyrocarbonate
(DEPC) [Sigma D-5758, (0.1%, v/v)] and autoclaved.
As for all laboratory procedures described in this
article, gloves should be worn at all times to protect
both the operator and the experiment. This, too, prevents
the introduction of RNases and foreign RNA or
DNA in the reverse transcriptase (RT) and polymerase
chain reaction (PCR).
1. For RNA Isolation and Quantification
TRI Reagent (Sigma T-9424), chloroform (Sigma
C-2432), isopropanol (Sigma 1-9516), ethanol [Sigma
E-7037; prepare as 75% (v/v) in H2
micropipettors, tips, Eppendorf tubes, etc., spectrophotometer
(e.g., SpectraMax Plus plate reader,
Molecular Devices), and quartz cuvettes or Nanodrop
(ND-1000; Labtech Int. Ltd.)
2. For RT and PCR Reactions
Southampton, UK); MMLV-RT enzyme (200U/µl)
(Sigma M-1302); 5X buffer (Sigma B-0175); dithiothreitol
) (Sigma D-6059); RNasin (40U/ml)
(Sigma R-2520); dNTPs (10mM
each of dATP, dCTP,
dGTP, and dTTP for RT; 1.25 mM
each for PCR) (Sigma
) (Sigma M-8787), Taq DNA
polymerase enzyme (5U/µl) (Sigma D-6677); 10X
buffer (Sigma P-2317); target primers and internal control primer (see Table II) (for further details on
primer selection criteria, see O'Driscoll et al.
3. Gel Electrophoresis
Amplified products are analysed by gel electrophoresis
(see Section IIIB).
: Several of the stains and other reagents used
(e.g., DEPC) should be handled with caution as they
are known or thought to be toxic, carcinogenic, and/or
A. Light and Fluorescence Microscopy
Detection of Apoptotic Cell Morphology
As described previously, a cell undergoing apoptosis
proceeds through various stages of morphological
change (Wilson and Potten, 1999). Light microscopy
and fluorescence microscopy are probably the simplest
and most basic techniques by which such apoptotic
cell death can be investigated. A broad range of stains
and dyes are available to assist in the assessment of
nuclear morphology. For light microscopy, the nuclear
stain haematoxylin is used frequently (often with eosin
as a counterstain). The most commonly used DNA
nucleic acid-reactive fluorochromes for UV light fluorescence
microscopic analysis of fixed (porated using,
e.g., methanol or ethanol) cells include DAPI, PI,
Hoechst 33258 and 33342, and AO. (As mentioned previously,
PI, DAPI, and Hoechst are also very useful for
assessing the membrane permeability of cells).
Using LM, cells dying by apoptosis are identified
by their reduced size, cell membrane "blebbing/
budding," and loss of normal nuclear structural features-
nuclear fragmentation and chromatin condensation.
In contrast, characteristic features of necrotic
cell death include cell and nuclear swelling, cytoplasmic
vacuolisation, patchy chromatin condensation,
and plasma membrane rupture.
If assessing adherent cell types, the cells may be
grown directly on coverslips (plated in Petri dishes).
For suspension cells, cytospins are generally prepared.
For both monolayer and suspension cultures, it is
important to consider cell concentration. Fifty to 70%
confluency of the area being analysed is generally considered
optimalmif the cells are more confluent, overlapping
may occur, which may hinder analysis; if cells
are too sparse, an accurate examination may not be
achievable. Typically, 1-2 × 105
cells suspended in
100µl PBS containing 1% (w/v) fetal calf serum (FCS)
should be cytocentrifuged onto microscope slides using a low-speed, short centrifugation (e.g., 600-
1000rpm for 2-4min); however, this should be
optimised as relevant for cells being analysed.
For LM only. 0.1M haematoxylin
: In a fume hood,
dissolve 1 g haematoxylin (BDH 34242) in 1 litre distilled
water, boil for 5 min, remove from heat, and add
0.2 g sodium iodate (Sigma S-4007). After 10 min, in the
order listed, add 50g aluminium potassium sulphate
(Sigma A-7167), 1 g citric acid (Sigma C-2404), and
50g chloral hydrate (Sigma C-8383), allowing each to
dissolve completely prior to adding the next. The complete
solution is stable for approximately 3 months at
For FM only
. Fluorescent stains should be stored
in light-proof containers to prevent quenching.
- DAPI: Prepare stock at 5 mg/ml in methanol (store
at -20°C for up to 3 months). Prior to use, dilute
1:10,000 in PBS, pH 7.4.
- PI: Prepare stock at 2mg/ml in PBS, pH 7.4 (store
at -20°C for up to 3 months). Prior to use, dilute
1:1500 in PBS.
- Hoechst 33258 and 33342: Prepare stock at
100µg/ml in PBS, pH 7.4 (store at -20°C for up to
3 months). Prior to use, dilute 1:10 in PBS.
- AO: Prepare stock at 2mg/ml in PBS, pH 7.4 (store
at -20°C for up to 3 months). Prior to use, dilute
1:400 in P BS.
: When working with fluorochromes, minimise
exposure to light at all stages of preparation.
B. Gel Electrophoresis for DNA Ladder
- Fix cells in ice-cold methanol for 5-7 min.
- Allow to air dry.
- Using a grease pen, encircle area of cells for analysis.
Typically a circle of 10 mm diameter or smaller
is drawn (to contain solutions and to minimise
volumes of solutions, antibodies, etc., required).
- Stain cells with 0.1% hematoxylin (for LM) or DAPI,
PI, Hoechst, or AO (for FM) for 3-5 min.
- Wash in distilled H2O (for LM) or PBS (for FM).
- Mount coverslip on slide using an aqueous-based
mountant medium and fix in place (clear nail
varnish may be used to secure coverslip).
- Immediately analyse and photograph samples by
fluorescence microscopy. Slides cannot be stored
long term as fluorescence quenches. However,
storing at 4°C in the dark will prolong the life span
of the signal. Haematoxylin-stained slides may be
Activation of endogenous Ca2+
- and Mg2+
dependent nuclear endonuclease(s) cleaves DNA
into discrete fragments, initially into 300- to 50-kb
fragments and subsequently into 180-bp fragments. In
brief, for DNA fragment detection by gel electrophoresis,
DNA is extracted from cells and loaded
onto a 1.5% agarose gel containing ethidium bromide.
The DNA fragments form a characteristic "ladder"
pattern resulting from multiples of a 180-bp DNA
subunit, representing DNA of the size of individual
nucleosomes and oligonucleosomes. [Nuclear DNA
damage resulting in death by necrosis, however, is
random and results in smears on a gel (Ramachandra
and Studzinski, 1995).]
- Lysis solution: 50mM Tris-HCl (pH 8.0), 20mM EDTA, 10mM NaCl, and 1% (w/v) SDS. A 50-ml
aliquot stock may be prepared and aliquotted in 2-ml
volumes (store at -20°C for up to 3 months).
- TBE buffer: 1X TBE consists of 10.8g Tris base,
5.5 g boric acid, and 4 ml 0.5 M EDTA (pH 8.0) made up
to 1 litre with ultrapure water. It is advisable to prepare
as a 10X concentrate and store at room temperature for
up to 3 months.
- RNase A: Prepare stock of 200µg/ml and store at
-20°C (for up to 3 months) to use at a final concentration
- Proteinase K: Prepare stock of 1 mg/ml and store
at -20°C (for up to 3 months) to use at a final concentration
- Ethidium bromide: Prepare a 10 mg/ml stock of
ethidium bromide by adding 1 g of ethidium bromide
to 100 ml of H2O. Stir on a magnetic stirrer for several
hours to ensure that the dye has dissolved. Wrap the
container in aluminium foil or keep in a dark bottle.
Store at room temperature. Note: It is important to consider
that ethidium bromide is a potential carcinogen.
Due caution should be taken when preparing ethidium
bromide stock, adding ethidium bromide to gel, disposing
of exposed pipette tips and ethidium bromidecontaining
- DNA loading buffer: DNA loading buffer may
be prepared at a 6X concentrate by mixing 1 mg/ml
bromphenol blue, 1 mM EDTA, and 50% glycerol (v/v)
in ultrapure H2O. Store at room temperature (should
be stable for up to 12 months).
- Harvest cells for analysis, wash with PBS, and
pellet by centrifugating for 5 min at 1000rpm.
- Lyse cell pellet by adding a volume of lysis
solution containing 50mM Tris-HCl (pH 8.0), 20mM EDTA, and 10mM NaCl, 1% (w/v) SDS and incubating
for 10min at 37°C (the cell number used and the
volume of lysis solution added must be optimised for
each cell type being analysed because if the sample
is too viscous the resulting gel resolution may be
reduced). It is important to ensure at this stage that the
pellet has broken up. This may be assisted by "flicking"
the tube gently.
- Add RNase A (final concentration of 20µg/ml) to
the lysate and incubate for 60min at 37°C.
- Add proteinase K (final concentration of
100µg/ml) to the lysate and incubate for 4h at 37°C. Lysates may then be loaded directly onto an agarose
gel (as described later). If clear bands are not detected
using whole cell lysates, DNA may be purified from
cell lysates at this stage using standard phenol/chloroform/
isoamyl alcohol procedures (see Sambrook
and Russell, 2001).
- During the incubation steps, dilute a stock of 10X
TBE to 1X TBE to use in preparation of a 1.5% agarose
gel. 1X TBE buffer is also prepared for use as running
- Melt and pour 1.5% agarose gel in a horizontal
gel support (taking safety pre cautions when working
with molten agarose). Insert the comb and allow the
gel to solidify.
- Add loading buffer to lysates (to resulting in a
final 1X concentration of loading buffer), "flick" tubes,
and centrifuge briefly.
- Load 10- to 50-µl samples to the gel wells and run
the gel in 1X TBE at 75V for approximately 1.5h at
room temperature. Molecular weight markers should
be loaded to allow lysate band sizes to be estimated
- Stain gel with 5µg/ml ethidium bromide in 1X
TBE for 30min.
- Place the gel on a UV transilluminator box to
visualise (and photograph) resolved DNA fragments
in a ladder pattern. (Note: Wear appropriate safety gear
to ensure that eyes and skin are not exposed to UV
TUNEL is a cytochemical method suitable for
analysis of apoptotic DNA fragmentation in individual
cells. This technique involves in situ
labelling of the 3'-OH ends of fragmented DNA in
fixed, permeabilised cells with either enzyme (phosphatase
or peroxidase) or fluorochrome-tagged
deoxynucleotides using terminal deoxynucleotidyl
transferase (TdT). (Note: If DNA polymerase I is used instead of TdT, the method is termed in situ nick
translation.) If fluorescence labelling is chosen, the
fluorescein-12-dUTP-labelled DNA can then be visualised
directly by fluorescence microscopy to give
qualitative results. If facilities are available and quantitative
results are required, flow cytometric analysis
may be used.
- Fixation solution: 4% paraformaldehyde in PBS
(pH 7.4) is prepared immediately before use. To help
dissolve the paraformaldehyde, this solution may be
placed on a magnetic stirring box and heated very
gently. Paraformalydehyde should only be prepared
and used in a fume hood.
- Permeabilisation solution: 0.1% Triton X-100 in
- Grow cells for analysis as a monolayer on glass
slides. Alternatively, cytospin preparations of cells
may be used.
- Remove culture medium, wash cells twice with
PBS, and allow to air dry.
- Fix cells by immersing slides in a Coplin jar
containing fixation solution, i.e., freshly prepared 4%
paraformaldehyde in PBS (pH 7.4), for 25 min at 4°C
- Rinse the slides by immersing in PBS for 5 min at
room temperature (two times). It is very important that
the slides are not allowed to dry out during the following
- Gently tap off excess PBS, cover cells with 100 btl
of equilibration buffer, and incubate for 5-10min at
- While incubating in equilibration buffer (step 5),
prepare the TdT incubation buffer by adding 45µl
equilibration buffer + 5µl nucleotide mix + 1 µl TdT
enzyme per sample (prepare as a master mix, depending
on the number of samples being analysed). As a
negative control, TdT may be eliminated from some
samples and replaced with an equal volume of ultrapure
H2O. It is important to keep the nucleotide mix
and TdT incubation buffer on ice and to ensure that all
steps from now on be protected from direct light.
- Following equilibration, carefully blot off excess
equilibration buffer and add 50µl of TdT incubation
buffer to the cells. Cover with plastic coverslips to
ensure even distribution of the TdT incubation buffer
and to prevent slides from drying out.
- Place slides in a humidified chamber (a suitably
sized flat-bottomed plastic box containing a layer of paper towels soaked in water is ideal for this purpose).
Cover with aluminium foil to protect from light and
incubate for 60min at 37°C to allow the "tailing" reaction
- To terminate the reaction, 20X SCC buffer should
be diluted to a 2X SCC solution with ultrapure water
and placed in a Coplin jar, the coverslips removed
from the slides, and the slides immersed for 15 min at
- To remove unincorporated fluorescein-12-
dUTP, the slides should be washed by placing in PBS
for 5min at room temperature (repeat wash two
- If desired, slides may be stained at this stage by
immersing in Coplin jars containing freshly prepared
PI solution (see preparation details given earlier) and
incubating for 15 min at room temperature. Following
this, wash slides in ultrapure water (3 × 5 min at room
- Mount slides using Vectashield and glass
- Immediately analyse and photograph cells by
fluorescence microscopy (520 mm filter for fluorescein
and >620 mm filter if propidium iodide has been
included. Slides cannot be stored long term as fluorescence
quenches. However, storing at 4°C in the dark
may prolong the life span of the signal.
Expression of apoptosis-related mRNAs may be
analysed using RT-PCR methods. This section
describes the use of "basic" RT-PCR, but it is important
to be aware that depending on the requirements
of the study and the resources available, RT-PCR may
be used to indicate the presence or absence of transcripts
of interest or it may be developed as a semiquantitative
or a quantitative level using real-time
RNA may be isolated from cells using the procedure
described by Chomczynski and Sacchi (1987).
However, there are now a number of less laborious,
commercially available methods, including the use of
TRI reagent (Sigma), as described here. The RT procedure
detailed involves use of the MMLV-RT (Sigma)
enzyme for cDNA synthesis and the PCR uses Taq
DNA polymerase (Sigma). Again, however, there are
other reverse transcriptases and DNA polymerase
enzymes available that may be equally suitable.
Primers suitable for the analysis of human bcl-2,
bag-l, bax-α, mcl-1, galectin-3 (designed in our laboratory),
and survivin are included in Table II, as examples
of apoptosis-related gene transcripts that may be
desirable to analyse by RT-PCR. There are, of course,
many more mRNAs involved in apoptotic pathways that should be considered. The methods described
here may be adapted by using relevant primers for the
amplification of other apoptosis-related cDNAs [for
information on primer design and selection criteria,
see O'Driscoll et al.
Table II also includes primer sequences for coamplification
of cDNA derived from an endogenous
"housekeeping" mRNA, β-actin, as control. Inclusion
of such primers serves to indicate that the RT and PCR
reactions have been performed successfully; if
performing semiquantitative PCR, this allows the
apoptosis-related gene transcript results to be normalised
relative to a control that (generally) should be constant.
RNA Isolation and Quantitation
Typical RT Reaction
- Pellet cells and wash with PBS (three times).
- In a 0.5-ml Eppendorf tube, lyse cells in TRI
reagent; 1 ml TRI reagent is suitable to lyse approximately
0.5-1 × 107 cells.
- RNA may be isolated immediately or lysates
stored at -80°C for up to 1 month.
- Incubate lysates at room temperature for 5 min.
- Add 200 µl chloroform per 1 ml TRI reagent used,
shake samples vigorously for 15s, and incubate
at room temperature for 15 min.
- Centrifuge at 12,000g, at 4°C for 15 min. Following
this, the RNA will be contained in the upper
aqueous phase, below which are the DNA-containing
interface and the protein-containing organic phase.
- Transfer the RNA-containing aqueous phase
to a clean 0.5-ml Eppendorf tube, add 0.5ml isopropanol,
mix, and incubate at room temperature for
- Centrifuge at 12,000g at 4°C for 10min to pellet
- Remove supernatant (carefully, to prevent
disturbing RNA pellet), wash RNA with 1 ml 75%
ethanol, vortex for 5s, and centrifuge at 7500g at 4°C for 5 min.
- Remove ethanol, air dry pellet briefly, and
resuspend in 25-50µl DEPC-H2O. (Ensure that the
pellet does not dry completely, as this decreases its solubility
greatly. The solubility of RNA can be improved
by heating to 55-60°C with intermittent vortexing or
by passing the RNA through a pipette tip, if necessary).
Store RNA at -80°C.
- Quantify RNA spectrophotometrically at 260
nm and 280nm. The A260/A280 ratio of RNA is approximately
2. [Partially solubilised RNA has a ratio <1.6
(Ausubel et al., 1991).]
Typical PCR Reaction
- To a 0.5-ml Eppendorf tube add 1µl
oligo(dT)12-18mer (1 µg/µl), 1 µl RNA (at 1 µg/µl), and 3µl DEPC-H2O. Mix gently, incubate at 70°C for
10 min, and chill on ice. [Note: For survivin analysis, to
avoid coamplification of the homologous cDNA for
effector protease receptor-1 (EPR-1), instead of including
the oligo(dT) primer, use 250ng of the survivinspecific
RT primer 5' AGGAACCTGCAGCTCAGA 3'.]
- Add the following reagents: 4µl 5X buffer, 2µl
(100 mM) DTT, 1 µl (40 U / µl) RNasin, 1 µl (10 mM each)
dNTPs, and 6 µl (200 U/µl) MMLV-RT. Mix.
- Incubate at 37°C for 1 h, followed by 95°C for
IV. SELECTION OF A SUITABLE
METHOD FOR APOPTOSIS
- To a 0.5-ml Eppendorf tube add the following
reagents: 24.5 µl H2O, 5 µl 10X buffer, 3 µl (25 mM)
MgCl2, 8µl (1.25mM) dNTPs, 1µl target forward
primer (250ng/µl) (see Table II), 1µl target reverse
primer (250ng/µl), 1µl control forward primer
(250ng/µl), 1µl control reverse primer (250ng/µl),
and 0.5 µl (5 U/µl) Taq polymerase enzyme. Mix gently.
Add 5µl cDNA (from RT reaction). Mix gently and
centrifuge very briefly to collect solution at bottom of
tube. (Note: Primer concentrations used may need to
be increased or decreased to optimise amplification.)
- Amplify on a thermocycler using the following
PCR cycle: 94°C for 2min; 30 cycles of 94°C for 30s,
relevant annealing temperature (Table II) for 30 s, 72°C for 30s; completion step of 72°C for 5min. Cycle
numbers may be reduced to prevent reaching the
plateau phase of PCR amplification if semiquantitative
analysis is required.
- Analyse RT-PCR products by agarose gel electrophoresis
(see Section III,B).
There are many factors to be considered when selecting
a suitable method for investigating apoptosis.
These factors include the cell type being analysed, the
nature of the cell death inducer, the stage of cell death,
the information required from the study (e.g., whether
information on single cells or on the cell population,
as a whole, is required), and the resources available
(e.g., where or not access is available to a fluorescence
microscope, flow/cytometer, thermocycler, etc.). To
assist with selection, Table III summarises the "advantages"
and "disadvantages" of the procedures detailed
in this article. To form a more extensive understanding
of the events occurring within cells, it is advisable,
whenever possible, to investigate cell death using
more than one technique.
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