the pros and cons of apoptosis assays for use in …...review the pros and cons of apoptosis assays...

Review

The Pros and Cons of Apoptosis Assays for Usein the Study of Cells, Tissues, and Organs

Michiko Watanabe,1* Midori Hitomi,1 Kathy van der Wee,2 Florence Rothenberg,2

Steven A. Fisher,2 Robert Zucker,3 Kathy K.H. Svoboda,4 Edie C. Goldsmith,5

Kaisa M. Heiskanen,6 and Anna-Liisa Nieminen6,7

1Department of Pediatrics (UHC), Case Western Reserve University, Cleveland, OH 44106, USA2Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA3Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory,

Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA4Department of Biomedical Sciences, Texas A&M Health Science Center, Baylor College of Dentistry, Dallas, TX 75246, USA5Department of Developmental Biology and Anatomy, University of South Carolina School of Medicine,

Columbia, SC 29209, USA6Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106, USA7CWRU/University Hospitals Ireland Comprehensive Cancer Center, Cleveland, OH 44106, USA

Abstract: Programmed cell death or apoptosis occurs in many tissues during normal development and in the

normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and

disease. Increased interest in apoptosis and cell death in general has resulted in the development of new

techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it

appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the

strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Key words: cell death, confocal microscopy, immunohistological techniques, mitochondria, programmed cell

death

INTRODUCTION

In the past 10 years, there has been an extraordinary in-

crease in research aimed at understanding the mechanisms

and processes that underlie programmed cell death or apop-

tosis. This increased interest in apoptosis has arisen from

the realization that this particular form of cell death occurs

during both normal development and disease progression

and treatment ~Thompson, 1995; Anversa et al., 1997; Yaoita

and Nakajima, 1997; van den Hoff et al., 2000!. Apoptosis

can be characterized by an orchestrated series of events in

cultured cells beginning with plasma membrane blebbing

followed by extension of echinoid protrusions, plasma mem-

brane blistering, and, ultimately, cell lysis ~Collins et al.,

1997!. In tissues, the apoptotic cells or cell fragments are

phagocytosed before cell lysis with little or no immune

response.

Apoptosis is initiated by multiple triggers and regulated

by a complex network of molecules ~reviewed in Blanken-

Received July 30, 2001; accepted March 6, 2002.

Kaisa M. Heiskanen is now at the Department of Biology, Laboratory of Animal

Physiology, University of Turku, Finland.

*Corresponding author. E-mail: [email protected]

Microsc. Microanal. 8, 375–391, 2002DOI: 10.1017/S1431927602010346 MicroscopyAND

Microanalysis© MICROSCOPY SOCIETY OF AMERICA 2002

berg et al., 2000; De Laurenzi and Melino, 2000; Matsuyama

and Reed, 2000; Nicholson, 2001!. Pathways leading to

apoptosis are generally separated into two interconnected

pathways, one requiring the activation of death ligands and

receptors ~Schulze-Osthoff et al., 1998; Ashkenazi and Dixit,

1999! and another involving the mitochondria ~Waterhouse

and Green, 1999; Shi, 2001!. There is now evidence that the

two are linked and that molecules in one pathway can

influence the other. Whatever the triggering signal, the

apoptosis pathways convene on the same terminal pathway

that is initiated by the cleavage activation of Caspase-3.

Most of the commonly used apoptosis assays detect the later

events of apoptosis that occur in the terminal apoptotic

cascade ~Fig. 1!.

Apoptosis is often contrasted with necrosis. Necrosis is

associated with the explosion of cells including a rapid

degradation and increase in the permeability of the plasma

membrane, swelling of the mitochondria, and a subsequent

immune response. In contrast, apoptosis can be considered

to be the implosion of cells into compact membrane-bound

packages termed “apoptotic bodies” that are phagocytosed

by macrophages or other surrounding cells. In apoptosis,

the immune response is minimally activated although excep-

tions have been noted ~Bowen-Pope and Schaub, 2001!.

Apoptosis has also been defined as cell death involving

caspase activity and DNA fragmentation ~Haunstetter and

Izumo, 1998!. Cells also die with atypical characteristics

that make it difficult to put them in either category. New

categories have been proposed including autoschizis ~La-

tour et al., 2000; Taper et al., 2001! and parapoptosis ~Spe-

randio, 2000!. A discussion of how to categorize the various

forms of cell death and terms to refer to these cell deaths

continues ~Majno and Joris, 1995; Levin, 1999; Wyllie and

Golstein, 2001!.

One of the challenges of assaying for apoptosis is the

speed at which cells die and disappear. The time from

initiation of apoptosis to its completion can be as short as

2–3 hr ~Bursch et al., 1990a, 1990b!. In a system that is

being investigated for apoptosis for the first time, assaying

too early or too late can result in false negative findings

because the cells can die and disappear without a trace

within a few hours. Collection of data from multiple high-

resolution time points would need to be assayed to ensure

that this oversight does not occur. Another challenge is that

apoptosis can occur at low frequency or in specific sites

within cultures, tissues, or organs. An example is found in

studies of human carcinoma cells used to describe the

morphological sequence of events in apoptosis. It was dem-

onstrated that the apoptotic time course ~12–24 hr! was

dependent upon the induction mechanism and not all cells

responded at the same time resulting in asynchronous apop-

totic cell cultures ~Collins et al., 1997!. In these cases, the

ability to survey large areas rapidly is valuable.

For this review, a subset of the available apoptosis and

other cell death assays was chosen for discussion. Most are

commonly used apoptosis detection techniques and have

been used extensively by the authors. Many of the authors

are studying cell death in developmental systems. Original

findings using these methods are presented in some sections.

TRANSMISSION ELECTRON MICROSCOPY~TEM!

Apoptosis was a term originally used to refer to a cellular

state defined by morphological criteria ~Kerr et al., 1972;

Wyllie et al., 1980!. Categorization of a dying cell as apop-

totic is considered irrefutable if the cells meet ultrastruc-

tural criteria obtained by standard transmission electron

microscopy. The characteristics of an apoptotic cell as as-

sessed by this method are: ~1! intact cellular membranes

even late in the cellular disintegration, ~2! dark and dense

cytoplasm and nucleus, ~3! blebs at the cell surface, ~4! large

clear vacuoles, ~5! disorganized cytoplasmic organization,

and ~6! nuclear fragmentation. The nucleus of an apoptotic

Abbreviations

ACM actin cortical mat

AM acetoxymethyl

Apaf1 apoptosis protease-activating factor-1

ECM extracellular matrix

LSCM laser scanning confocal microscopy

LTR Lysotracker Red

MB methylene blue

MPT mitochondrial permeability transition

NR neutral red

PF paraformaldehyde

PS phosphatidylserine

ROS reactive oxygen species

TB toluidine blue

TEM transmission electron microscopy

TMRM tetramethylrhodamine methylester

TUNEL Terminal dUTP Nick End-Labeling

376 M. Watanabe et al.

cell can also become “marginalized.” In this situation, dark

and dense nuclear material appears to congeal at the nuclear

membrane. Apoptotic cells within a tissue have few if any

cell–cell junctions connecting them to their neighbors and

often appear pulled away from the neighboring cells as they

round up. At late stages, the cells break up into smaller sized

particles. These apoptotic cell fragments or bodies can often

be observed engulfed by surrounding cells such as embry-

onic cardiomyocytes ~Hurle et al., 1978! and adult vascular

smooth muscle cells ~Bennett et al., 1995! or cells special-

ized to be scavengers such as macrophages and Kuppfer cells

of the liver. In some cases, TEM allows simultaneous identi-

fication of cells undergoing apoptosis and the cell type. At

early apoptosis stages the cell type can be identified by the

presence of characteristic features such as striated myo-

fibrils in the case of cardiomyocytes ~Fig. 2! or skeletal

muscle cells ~Borisov and Carlson, 2000!. Immunostaining

for cell-specific antigens will also identify cell types. Unfor-

tunately cells that can be assayed by the classic ultrastruc-

tural criteria are at a late stage in their demise and often lack

the structures or antigens that would identify the cell type.

One of the main disadvantages of TEM is that only a small

region can be observed at one time. This makes surveys and

counts of apoptotic cells by TEM tedious and time-

consuming. Another disadvantage is that preparation of

tissues for TEM requires harsh processing conditions such

as exposure to alcohol, organic solvents, and resins that are

cured at high temperatures. The processing can destroy

antigens or enzymes used to identify specific cells.

STAINED RESIN SECTIONS

Resin-embedded tissues can be sectioned at ;1 mm and

stained with toluidine blue ~TB! or methylene blue ~MB! to

reveal intensely stained apoptotic cells when observed by

standard light microscopy ~Fig. 3!. The tissue and cellular

details are well preserved by this technique. Surveys of large

regions can be done quickly and, once a potentially interest-

ing region of apoptotic cells is identified at the light micro-

scope level, the tissue can be trimmed and sectioned for

more detailed TEM observations. One disadvantage is that

this assay will underestimate the extent of apoptosis occur-

Figure 1. Most of the apoptosis assays detect cellular changes late in apoptosis. The participants in initiation and

execution of apoptosis are shown in white letters on a black background. Assays for earlier events in apoptosis are

presented in the small black squares. The late events in apoptosis are listed in the rectangular box with the

corresponding detection techniques next to them in parentheses. AnxV 5 Annexin V, Apaf1 5 apoptosis protease-

activating factor-1, Death L 5 death ligand, Death R 5 death receptor, dex 5 dexamethazone, MPT 5 mitochondrial

permeability transition, UV 5 ultraviolet light. Other abbreviations are listed on the second page of this article. Diagram

by Colleen Kurzawa.

Apoptosis Assays 377

Figure 2–6. ~See captions on facing page.!


ring compared to TEM because smaller apoptotic bodies

will not be detected. Another disadvantage is that healthy

cells with large dense intracellular granules can be mistaken

for apoptotic cells or cell fragments. There are also a few

cases where perfectly healthy cells with condensed nuclei

such as lymphocytes or those undergoing mitosis may be

mistaken for apoptotic cells. Another disadvantage similar

to that for TEM is that harsh preparation conditions usually

destroy enzyme activity and antigenic sites thus preventing

postembedding enzyme assays or immunostaining in most

cases.

DNA LADDERING

One of the accepted indicators of apoptosis is the fragmen-

tation of DNA that results in a characteristic “DNA ladder”

where each band in the ladder is separated in size by

approximately 200 bp. The absence of a DNA ladder does

not eliminate the potential that cells are undergoing apop-

tosis because DNA fragmentation occurs late in the apop-

totic process ~Collins et al., 1997!. When cells undergo

apoptosis, endonucleases are activated which cut the DNA

into fragments at sites between the nucleosomes ~for review,

see Zhang and Xu, 2000!. This results in regularly sized

DNA fragments that separate as regularly spaced bands on

an agarose gel. In necrosis, the DNA fragments are ran-

domly cut and dispersed rather than concentrated in

membrane-bound packages. The assay for DNA laddering is

useful for tissues or cell cultures that have high numbers of

apoptotic cells per tissue mass, but not for those with low

numbers.

TUNEL

Labeling DNA fragments is a well accepted and widely used

histological assay for detecting apoptotic cells. This tech-

^

Figure 2. Transmission electron microscopy ~TEM! reveals ultrastructural features of an apoptotic cardiomyocyte in

the embryonic chicken outflow tract myocardium, dark cytoplasm, and clear large vacuoles or vesicles ~arrowheads!.

The cell type was determined by the presence of bundles of myofilaments in the cytoplasm. The diameter of this

apoptotic cell or cell fragment at its widest point is 4 mm. It has been engulfed by a healthy cardiomyocyte.

Figure 3. Methylene blue-stained resin section ~MB! of the embryonic chicken outflow tract. A focus of dying cells was

detected at the distal rim within the outflow tract myocardium in sagittal section. The apoptotic cells and bodies stain a

dark blue ~between black arrows!, but with heterogeneity in size and intensity. The smaller homogeneous blue granules

are found in healthy cells.

Figure 4. Annexin V-stained ~AnxV! cardiomyocyte in the embryonic chicken proximal outflow tract myocardium. The

striated myocyte marker, a monoclonal antibody MF20 that recognizes an isoform of myosin heavy chain, stains the

cytoplasm green ~Alexafluor-488!. The Annexin V-biotin was detected on the plasma membrane of this cell using

avidin-Texas Red. The confocal image was taken with a z-section of 0.8 mm. The cell is approximately 17 mm long.

Figure 5. Comparison of Annexin V ~A! and PhiPhiLux ~B! staining on cultured corneal epithelial from 8-day chicken

embryos. Annexin V ~A! was tagged with Alexafluor 488 ~green! and stains with the characteristic ring pattern of plasma

membrane markers. The same section was costained for actin ~red!. In contrast, the PhiPhiLux ~B! stains the cytoplasm

~green!.

Figure 6. Whole-mount stage 25 quail embryo ~A! in frontal view to show LTR staining ~band of intensely bright red

particles! at the base of the cardiac outflow tract ~oft!. rv 5 right ventricle, lv 5 left ventricle. Hearts are ;1 mm wide at

this stage. Embryonic day 9 mouse ~B! cultured with 250 mM hydroxyurea in vitro for 24 hr ~starting on day 8! and

stained with LTR. LTR staining ~white particles! was increased relative to the control and was detected throughout the

embryo except in the heart ~hrt!. This is a view of the right side of the embryo with the dorsal surface to the left, the

ventral to the right, and the cranial portion is to the top. The heart is 1 mm in diameter. The image is a maximum

projection of 25 individual confocal sections.


nique assays endonuclease cleavage products described in

the last section, the regularly sized DNA fragments that

accumulate and remain concentrated in packages sur-

rounded by intact nuclear membranes. These highly concen-

trated DNA fragments are detected by using terminal

transferase to add labelled nucleotides to the 3' end of DNA

fragments. The procedure is called the Terminal dUTP Nick

End-Labeling ~TUNEL! reaction ~Modak and Bollum, 1972;

Gavrieli et al., 1992!. The dUTP can be labeled with a

variety of probes to permit detection by fluorescence micros-

copy or colorimetric assay. One commercially available kit

uses digoxigenin-labeled nucleotides. The digoxigenin is

detected with antidigoxigenin antibodies tagged with either

a fluorescent marker or peroxidase. The peroxidase label is

visualized by a colorimetric assay such as one using diami-

nobenzidine ~DAB! that results in dark brown precipitates

at the site of positive staining. TUNEL staining typically

requires some form of sample pretreatment, either using

proteinase K or microwaves, to improve sensitivity and the

inclusion of a blocking agent, such as bovine serum, to

reduce background ~Labat-Moleur et al., 1998!. The assays

are available as kits from a number of companies.

One disadvantage of TUNEL is cost. Both the enzyme

and antibody to digoxigenin are expensive. Therefore, this

assay may not be the preferred method for large-scale

analyses requiring the staining of many sections. In addition

to the expense, the TUNEL assay has other limitations. It is

not clear how many DNA breaks are necessary to yield a

positive signal by either TUNEL or the hairpin oligonucleo-

tide method described below ~Schaper et al., 1999!. The

method of fixation and pretreatment can also affect the

detection of DNA strand breaks ~Negoescu et al., 1996!.

Several studies have shown that TUNEL staining cannot

always distinguish apoptotic and necrotic cells ~Grasl-

Kraupp et al., 1995; Schaper et al., 1999! and sometimes this

technique falsely identifies cells in the process of DNA

repair. Additional studies have demonstrated that cells in

the process of active gene transcription can also yield false

positive TUNEL results ~Kockx et al., 1998!. Despite its

well-accepted status, TUNEL is not usually used as the sole

apoptosis assay because of these potential false positives and

is often paired with another assay.

The TUNEL reaction has been used in a number of

studies to evaluate the level of apoptosis during heart devel-

opment and failure. TUNEL staining was used to detect and

quantify the high levels of cardiomyocyte apoptosis associ-

ated with outflow tract shorting during heart development

in chicken embryos ~Watanabe et al., 1998!. In this study,

other assays ~caspase 3 activity, Annexin V! were used in

conjunction with TUNEL to support occurrence of apopto-

sis in that tissue. Two studies reported cardiomyocyte apop-

tosis levels during end stage heart failure in human cardiac

explants ranging from 0.2 to 35 TUNEL-positive nuclei per

100 cardiomyocytes ~Narula et al., 1996; Olivetti et al.,

1997!. The latter number is quite high and has caused

several investigators to question the accuracy of TUNEL

staining ~Schaper et al., 1999!.

Given the uncertainty with which TUNEL staining may

detect nonapoptotic nuclei, an additional method has been

developed that detects double-strand breaks in DNA intro-

duced during apoptosis. Oligonucleotides were synthesized

that were able to discriminate between DNA fragments

containing ends that were blunt or had 3' overhangs. Advan-

tage was taken of the fact that the enzyme~s! that produce

the DNA fragments found in apoptotic cells produce DNA

ends with free 5' phosphate groups capable of ligation to

foreign DNA. Initial experiments used Taq polymerase to

produce labeled ~fluorescent and nonfluorescent!, double-

stranded DNA fragments with a single 3' base overhang and

Pfu polymerase to generate labeled ~fluorescent and nonflu-

orescent!, double-stranded DNA fragments with blunt ends

~Didenko and Hornsby, 1996!. This method was tested on

both paraffin-embedded and frozen tissue samples with no

difference in sensitivity. By comparing the ligation of blunt

and 3' base overhang DNA probes to DNA fragments

generated through apoptotic mechanisms ~thymus treated

with either glucocorticoid or dexamethasone!, necrotic tis-

sue ~Wilms’ tumor!, and tissue with nonapoptotic DNA

damage ~peroxide treated liver, heat-denatured adrenal gland

sections, adrenal gland subjected to autolysis!, Didenko and

Hornsby demonstrated that double-stranded DNA frag-

ments with 3' base overhangs were characteristic of apop-

totic cells while blunt-end DNA fragments, detected to

lesser degrees in tissue with nonapoptotic DNA damage,

were also observed in these cells ~Didenko and Hornsby,

1996!. The inclusion of terminal deoxynucleotidyl transfer-

ase ~TdT! reactions demonstrated that TdT labeled all DNA

with a free 3' end irrespective of the origin of DNA damage.

These studies have now been expanded to include the

design and use of small ~;30 bp! biotin-labeled hairpin

oligonucleotides to detect double-strand DNA breaks

~Didenko et al., 1998!.

Other investigators have used this method to confirm

apoptosis detected with more traditional methods such as

TUNEL. This in situ ligation method was used to show that

TUNEL positive nuclei in cardiac myocytes from hearts in


the early stages of failure also stained positive for double-

strand DNA breaks ~Ding et al., 2000!. Oligonucleotide

hairpin probes have been used to correlate the activation of

caspase-3, an enzyme that cleaves, among other things,

Bcl-2, destroying its antiapoptotic activity while producing

c-terminal fragments which are proapoptotic ~Wolf and

Green, 1999!, with the presence of DNA damage in a rat

neuronal model of hypoxia-ischemia ~Zhu et al., 2000!. This

study used two other markers for DNA damage, TUNEL

and an antibody directed against single-stranded DNA, and

determined that the hairpin oligonucleotide demonstrated

the greatest degree of colocalization with caspase-3 activity.

In summary, TUNEL is a well-accepted assay to detect

concentrated DNA fragments and can be performed using

commercially available kits. The assay is time-consuming,

expensive, and can lead to false positive results. Some of the

pretreatments ~e.g., proteinase K! eliminate antigenicity and

the integrity of the tissues. As for any apoptosis assay, use of

a second assay based on a different principle is recom-

mended to confirm that what is detected is apoptosis.

ANNEXIN V

Annexin V, a 35–36 kDa protein, binds in the presence of

Ca21 with high affinity to the phosphatidylserine ~PS! resi-

dues exposed to the external plasma membrane surface of

cells dying by apoptosis ~Van den Eijnde et al., 1997a,

1997b; Blankenberg et al., 1999a, 1999b; Blankenberg and

Strauss, 1999!. Healthy cells also have these PS residues, but

manage to keep them all facing the internal or cytoplasmic

surface of the plasma membrane by the ATP-requiring

activity of enzymes called flippases or transmembrane trans-

porters ~for review of these enzymes see Daleke and Lyles,

2000!. When the cell begins to die, it quickly loses its ability

to keep these enzymes active and thus the PS residues

remain on the outside and are detected by Annexin V. The

everted PS serves as a signal that stimulates surrounding

cells to phagocytose the dying cell before the membranes

disintegrate. Annexin V is commercially available labeled

with either fluorescent markers for direct detection or for

indirect labeling using the biotin-avidin systems. The re-

agent must be injected or applied to live cells or tissues at

high concentration for at least 15–30 min before washing

and fixation. This makes it feasible for applying to cells in

culture and young embryos ~e.g., 6–12 somite chick em-

bryos! or injecting into the circulation of older embryos

~e.g., stage 30 chick embryos or 11–13 dpc mouse embryos;

Van den Eijnde et al., 1997a, 1997b; Watanabe et al., 1998!.

It would be very expensive to use for systemic injection into

adult animals even as small as mice. Another disadvantage is

that when injected into animals, Annexin V staining may

vary in intensity and pattern between animals depending on

the efficacy of the injection protocol. As with the other

apoptosis detection systems, it is possible to get positive

staining with Annexin V on certain cells that are not neces-

sarily dying. Subsets of healthy B-lymphocytes and intact

stimulated blood platelets express Annexin V on their sur-

faces ~Tzima et al., 1999; Dillon et al., 2001!.

Annexin V staining has an important advantage for

identifying cell types undergoing apoptosis, as was found in

the analysis of apoptosis in the embryonic myocardium. To

count the number of cardiomyocytes undergoing apoptosis

during development, sections were double-stained for MF20,

an antibody marker for proteins specific to striated myo-

cytes and TUNEL. When a cell was detected that was both

MF20 and TUNEL-positive, it was impossible to determine

whether it was an apoptotic cardiomyocyte containing its

own TUNEL positive nuclear material or a healthy cardi-

omyocyte that had just phagocytosed a TUNEL-positive

apoptotic body. This distinction can be made by using

Annexin V and scanning laser confocal microscopy with an

optical section that is less than 0.2–1 mm thick in the z-axis

~Fig. 4!. Another important advantage that Annexin V has

over TUNEL or vital dyes is that it allows detection of cells

at a slightly earlier phase in the apoptotic pathway ~Van den

Eijnde et al., 1997b! when antigens required for cell identi-

fication may still be intact.

CASPASE-3 ACTIVITY ASSAYS

Caspase-3 ~previously known as CPP32, YAMA, apopain! is

a cysteine protease activated in the terminal apoptosis cas-

cade. It is sequestered in the cytoplasm as a zymogen in its

inactive form and activated when cleaved by other caspases.

Its preferred sequence for protein cleavage is DEVD

~aspartate-glutamate-valine-aspartate! with an absolute re-

quirement for the terminal aspartate ~for review, see Nich-

olson and Thornberry, 1997!. The peptide DEVD-AFC,

when cleaved by Caspase-3, releases AFC, a fluorescent tag

that may be monitored in a fluorometer with the appropri-

ate excitation and emission settings. This method requires

homogenization of the cells of tissues to release the en-


zymes into solution. This step obviously destroys the integ-

rity of the tissue being assayed and precludes localizing the

event within the tissue or determining the cell type under-

going apoptosis. There is also a limitation in the size of the

tissues that can be assayed. For embryonic tissues, it is often

necessary to pool tissue dissections to get enough material

to detect the Caspase-3 activity. The advantage of this

method is that it allows rapid and consistent quantification

and is suitable for a time-course analysis for cells in culture.

It also works for complex tissues where several cell types are

present, but only one type undergoes changes in the level of

apoptosis. In the case of developing heart tissue in which

the epicardium, endocardium, and myocardium are all un-

dergoing apoptosis, the Caspase-3 assay allowed analysis of

the myocardial apoptosis only because it dominated at the

peak of apoptosis while the other tissues had fairly consis-

tent, low levels of apoptosis as assessed by TUNEL ~Wa-

tanabe et al., 1998!. We confirmed the dynamic changes in

the level of apoptosis in the myocardium by using the

TUNEL and Annexin V techniques in tissue sections taken

at the same stages.

The cell permeable peptide PhiPhiLux ~fluorescent-

fluorescent light; OncoImmunin, Inc.! was made to visual-

ize caspase-3 activity in living cells. This class of fluorogenic

protease substrates was used to determine if the corneal

epithelial sheets contained active caspase-3 and to compare

to Annexin V staining ~Fig. 5!. The PhiPhiLux molecule

contains the caspase-3-recognition sequence DEVD in a

bifluorophore-derivitized peptide that mimics the struc-

tural loop conformation present in the native protease

cleavage sites of globular proteins. Therefore, the com-

pound is not fluorescent unless it is cleaved by endogenous

caspase-3 ~Packard et al., 1997!. Corneal epithelial tissues

isolated without basal lamina ~2BL! respond to ECM using

an actin-dependent mechanism. The basal cell surface flat-

tens and the disrupted actin cortical mat ~ACM! reorganizes

in the presence of laminin, fibronectin, or collagen ~Sugrue

and Hay, 1981, 1986; Svoboda and Hay, 1987; Svoboda,

1992; Khoory et al., 1993! with distinct configurations in

the presence of different ECM molecules ~Svoboda et al.,

1999!. The loss of integrin-mediated cell–matrix attach-

ment induces apoptosis in many anchorage dependent cell

types. The corneal epithelial model used in this study main-

tains cell–cell attachments while separating the cell–matrix

attachment complex. A study was designed to determine if

disrupting the signal transduction pathways involved in

integrin-mediated actin reorganization increases apoptosis.

Briefly, corneal epithelia were isolated from 8-day chicken

embryos without the basal lamina and incubated overnight

with either control media or media containing 100 mg/ml

collagen I or 50 mg/ml fibronectin in the presence or ab-

sence of an inhibitor that blocks actin reorganization. In

the last hour of incubation, PhiPhiLux was added to the

media of the live tissues. Alternatively, tissues were incu-

bated in labeled Annexin V for 15 minutes. The tissues

were rinsed, fixed, and stained with Texas Red phalloidin.

Whole tissues were morphologically analyzed using a Leica

TSP SP2 confocal microscope for actin, Annexin V, and

FITC-PhiPhiLux ~Fig. 5!. Control tissues incubated with

or without fibronectin or collagen had low levels of PhiPhi-

Lux or Annexin V. In contrast, increasing doses of the

kinase inhibitor increased the staining, indicating that the

tissues were positive for Caspase-3 and Annexin V. It was

concluded that the Caspase-3 substrate and Annexin V

binding were more sensitive than other apoptosis detection

methods. In addition, incubating the cells with either sub-

strate did not disrupt staining for other proteins, such as

actin.

The advantages of these substances are that they can be

used on live cells and the PhiPhiLux penetrates cell mem-

branes and offers the caspase-3 a three-dimensional target.

In addition, both compounds can be visualized with confo-

cal microscopy and do not fade with laser excitation. The

disadvantages of PhiPhiLux are similar to that for Annexin

V. It is expensive and needs to be used at high concentra-

tions. Also, it may not penetrate into dense tissues or whole

embryos effectively.

VITAL DYES AND LYSOTRACKER RED

Apoptosis has been previously visualized in whole embryos

using the vital dyes, acridine orange ~AO!, Nile blue sulfate

~NBS!, or neutral red ~NR!. These dyes are acidophilic and

are concentrated in areas of high lysosomal and phagocyto-

sis activity. They have been used for a number of years even

before the term apoptosis was coined. These dyes would not

distinguish between lysosomes degrading apoptosis-

associated debris and other debris such as microorganisms.

For this reason, their use as apoptosis markers would have

to be validated using other apoptosis assays.

Acridine Orange is a cationic dye that binds DNA and

RNA by intercalating or electrostatic interactions. It has

green fluorescence ~emission maximum of 525 nm! when

bound to DNA and a red fluorescence ~emission shifted to


650 nm! when bound to RNA. Condensed chromatin can-

not be stained by AO. Under flow cytometry, apoptotic cells

exhibit lower levels of green fluorescence and higher levels

of red fluorescence. It can also be used on whole tissues and

embryos ~Buckiova et al., 1998; Farlie et al., 1999!. The

disadvantages are that it can be easily photobleached during

routine observation, it is a mutagen, and it is difficult to

maintain AO fluorescence during processing for analysis in

sections.

In developing embryos, Nile Blue Sulfate stains the

lysosomes and acidic cellular compartments that correlate

to regions where the apoptotic bodies are located ~Saunders

and Gasseling, 1962; Saunders, 1966; Menkes et al., 1979;

Hurle, 1988; Lumsden et al., 1991; Kotch et al., 1992; Kotch

and Sulik, 1992; Alles and Sulik, 1993; Gao et al., 1994; Allen

et al., 1997; Zakeri, 1998!. AO and NB colocalized in Dro-

sophila embryos. The distribution of these dyes closely

resembled that of pyknotic figures derived in plastic sec-

tions with TB staining ~Abrams et al., 1993!. From a sequen-

tial analysis of the same embryo, it was concluded that NBS

and AO staining in vivo was equivalent to TB staining after

fixation, and these dyes were indeed staining the apoptotic

cells/nuclei. It was also shown that AO and NBS were

detecting both free and phagocytosed cell corpses. NBS has

been shown by numerous investigators to be a suitable stain

to visualize apoptosis in the embryo or fetal limbs, as it

appears to stain the same specific regions that have been

designated as apoptotic by some other well-characterized

apoptotic assays such as TUNEL and the Annexin V assay

~Knudsen, 1992; Kotch and Sulik, 1992; Alles and Sulik,

1993; Zakeri et al., 1994; Van den Eijnde et al., 1997b;

Nakamura et al., 2000!.

Although NBS is valuable for quick, inexpensive analy-

sis of large numbers of specimens, the use of this vital stain

has limitations due to restricted dye penetration and poor

dye retention. It is also difficult to see the dye through

thicker tissue. NBS works well in whole mounts of small

embryos ~Buckiova et al., 1998; Farlie et al., 1999! or tissues,

but the dye staining is lost while preparing tissues for

sectioning. NBS and NR dyes can also label cells with

condensed granules that are not undergoing cell death.

LysoTracker Red ~LTR; Molecular Probes! acts in a

similar way to NBS by accumulating in the acidic intracellu-

lar compartments and regions where there is a high amount

of phagolysosomal activity resulting from the engulfment of

apoptotic bodies by adjacent cells ~Zucker et al., 1998, 1999,

2000a, 2000b!. LTR is a red fluorescent dye that excites with

a maximum at 568 nm and can be detected with standard

excitation and emission filters used to detect other red

fluorescent dyes such as rhodamine and Texas Red. It has

the added advantage of being an aldehyde fixable probe that

penetrates easily into tissues. The dye still maintains its

fluorescent characteristics in sample preparation procedures

that use 100% MEOH for dehydration and organic com-

pounds benzyl alcohol and benzyl benzoate ~BABB! for

clearing. These characteristics of LTR make it suitable to be

used with laser scanning confocal microscopy ~LSCM! to

provide three-dimensional imaging of cell death in tissue

that is as thick as 0.5 mm.

It has been previously shown that LTR has a similar

staining pattern as the other acidophilic dyes used in embry-

ology to stain apoptotic cells in the embryo and the limb

~Menkes et al., 1979; Kotch and Sulik, 1992; Zakeri et al.,

1994; Zucker et al., 1998, 1999; Nakamura et al., 2000!.

Whereas NBS and LTR were used for the morphological

identification of apoptotic bodies, TUNEL provided a

biochemical-based detection of apoptosis ~Gard, 1993; Allen

et al., 1997!. These data on lysomorphic dyes and Annexin

V/TUNEL biochemical data show that both morphological

and biochemical approaches visualize the induction of apop-

tosis. LTR staining ~Zucker et al., 1998, 1999, 2000a, 2000b!

and cell death labeling using other lysosomotropic dyes

~NBS, AO, and NR! were compared to other apoptosis-

specific biochemical assays ~TUNEL and Annexin V! to

show equivalency of cell death patterns with all the different

probes suggesting that they are staining the same regions. It

thus appears that LTR may directly correspond to biochem-

ical events represented by DNA strand breaks ~TUNEL! and

late stage Annexin V uptake in dead cells and may be

considered to be an indicator of apoptosis in tissue. This

LTR method has allowed visualization of apoptosis in whole

embryos, fetal limbs, and neonatal tissue using LSCM. LTR

staining in whole mounts of avian and murine embryonic

hearts ~Fig. 6A,B! has been used to identify in three dimen-

sions where and when apoptosis normally occurs and to

assess the effects of various reagents such as ETOH and

hydroxyurea ~Zucker et al., 1999! on apoptosis.

Technical difficulties associated with using LTR have to

be overcome if it is to be used as an accurate indicator of

apoptosis. These include the staining conditions of the dye.

This should be done in a tissue culture medium at 378C that

contains serum to protect and maintain the viability of the

tissue. The penetration of the dye into the tissue is crucial to

visualizing the regions of apoptosis in three dimensions.

Fixation should be done with an aldehyde ~4% paraformal-

dehyde @PF# or 1% glutaraldehyde! and the temperature


should be regulated to minimize fixation artifacts while

maximizing the sample fixation. PF was used at cold tem-

peratures and glutaraldehyde at all temperatures. Glutaral-

dehyde has the advantage of promoting excellent fixation,

but its major disadvantage was that it increased nonspecific

autofluorescence when using 488-laser excitation. By using

the 568 lasers to excite LTR, the autofluorescence was avoided.

LTR-stained intact embryos and hearts have been viewed

without further steps to visualize apoptosis close to the

surface of the samples ~within 300 mm from the surface!.

However, the clearing procedure is essential to visualize LTR

staining within thick tissue ~Klymkowsky and Hanken, 1991;

Gard, 1993; Zucker et al., 1998!. Extreme care must be taken

to remove all water from the samples and it is useful to

open fresh 100% MEOH in the final dehydration step. It is

also important to slowly change the percentages of MEOH

in the dehydration steps and BABB in the clearing steps to

minimize shrinking. BABB is damaging to plastic and glass

objectives; thus extreme care should be used to maintain the

samples in their holders and to prevent BABB from leaking

onto the surface of the slide. The use of air lenses is

recommended when viewing these samples so as to mini-

mize the risk of any contamination and potential damage of

expensive lenses with BABB. Finally it should be empha-

sized that LTR intensely stains acidic structures that may

not be associated with apoptosis as indicated by the distinct

nucleolar RNA staining in the normal oocyte ~Zucker et al.,

2000b!.

After the LTR-stained specimens are cleared in BABB,

the refractive index of the specimen becomes similar to the

surrounding BABB medium. Thus the specimen becomes

more transparent as there is a reduction of light scattering

and absorption effects. The result is that the laser can

penetrate the tissue with increased effectiveness. The whole

embryo can be observed with this approach. Individual

digital images of the embryo or other tissue are first ac-

quired by LSCM and then compiled into a whole three-

dimensional structure of the initial organism or tissue using

imaging software. The technique allows for the penetration

of light for over 0.5 mm with low power objectives and can

be increased using a two-photon laser confocal scanning

microscope. The LTR method of apoptosis detection does

provide an invaluable three-dimensional visualization of

embryonic apoptosis. This technique does have limitations,

which include a lengthy sample preparation using organic

compounds and the use of costly confocal equipment. An-

other limitation in using LTR is demonstrated by the obser-

vation that it can stain nonapoptotic regions in cells/tissues

that are acidic such as the nucleolus of an oocyte ~Zucker

et al., 2000b!.

Flow cytometry has been used to try to quantify apop-

tosis on a population of cells using a number of different

methods ~Darzynkiewicz et al., 1997!. It would be highly

desirable to make a similar type of assessment in tissues and

embryos. Previous attempts have been made to quantify

whole embryo cell death by detecting fluorescence intensi-

ties derived from whole embryos to provide a semiquantita-

tive assessment of apoptosis in teratogen-exposed embryos

~Gao et al., 1994; Lee et al., 1996; Ambroso et al., 1998!.

Preliminary data suggests that if conditions in the LSCM

are kept constant, it may be possible to evaluate apoptosis in

three dimensions with a LSCM using LTR fluorescent dye as

an indicator ~Zucker and Price, 1999!. There are many

variables that affect the analysis. Ideally it would be useful

to establish a threshold of pixel intensity, above which the

apoptosis index would occur. This apoptotic index will be

complex and subject to both biological errors of interpreta-

tion and technical errors in the acquisition of this data

using low power objectives. Due to the unpredictable nature

of apoptotic body formation and their subsequent engulf-

ment by neighboring cells, efforts to quantify apoptosis as a

function of fluorescence intensity can only be an approxi-

mation. However dose-dependent effects on fetal limb tis-

sue have been observed after treatment in vivo with the

chemotherapeutic drug 5-fluorouracil ~Lau et al., 2001!.

In summary, LTR has the advantages of being an inex-

pensive aldehyde fixable probe that penetrates easily into

tissues while being highly fluorescent even after harsh treat-

ments such as incubation in 100% MEOH ~dehydration! or

100% BABB ~clearing!. As a fluorescent agent, it can be used

with LSCM for double or triple labeling using other assays.

The properties of LTR allow analysis of whole-mount or-

gans, tissues, and embryos and reduce the need for sections

and three-dimensional reconstruction. The fluorescence is

very bright and resistant to quenching. This assay would not

work for cells that are known to have large acidic compart-

ments in their normal state such as macrophages or oocytes.

MITOCHONDRIAL ASSAYS

Mitochondria play an important role in apoptotic and

necrotic cell death. Depending on the stimulus, a variety of

mitochondrial dysfunctions can trigger the onset of cell

death. Assessment of mitochondrial dysfunction in living


cells in situ is important in understanding the sequence of

molecular and subcellular events producing cell injury and

death. LSCM creates submicron thin optical slices through

living cells and tissues and allows several mitochondrial

events to be monitored simultaneously in intact single cells

over time.

Several mitochondrial parameters, such as mitochon-

drial redox status, Ca21 increase, reactive oxygen species

~ROS!, the mitochondrial permeability transition, and mito-

chondrial depolarization can be monitored with LSCM in

single living cells. These events have been shown to occur in

apoptotic as well as necrotic processes. Oxidative stress can

induce oxidation of mitochondrial pyridine nucleotides that

can be visualized directly by ultraviolet LSCM of NAD~P!H

autofluorescence.

An increase of mitochondrial Ca21 can be measured by

the Ca21-indicating fluorophore, Rhod-2 ~Byrne et al., 1999!.

Rhod-2 AM has a net positive charge and is drawn electro-

phoretically into mitochondria where it is hydrolyzed to its

free calcium indicating form. Unlike other calcium indica-

tors, such as Fura-2 and Indo-1, Rhod-2 is not a ratiometric

probe and therefore quantitation of Ca21 concentrations

within cells is difficult. Rather, Rhod-2 monitors relative

changes in Ca21 concentrations.

A number of fluorophores can be used to measure

mitochondrial ROS generation. Nonfluorescent 2 ',7 '-

dichlorofluorescein is converted to highly fluorescent 2',7'-

dichlorofluorescein by hydroperoxides ~Nieminen et al.,

1997!. The probe that can be used to detect mitochondrial

superoxides is dihydroethidium. Dihydroethidium is oxi-

dized by superoxides to fluorescent ethidium. The oxidized

form of this probe, ethidium, binds to nucleic acids, thus

making the nuclei brightly fluorescent.

In isolated mitochondria, a wide variety of oxidant

chemicals induce increased nonspecific permeability of the

mitochondrial inner membrane to ions and solutes of mo-

lecular weight # 1500 Da. This mitochondrial permeability

transition ~MPT! causes mitochondrial swelling, membrane

depolarization, and uncoupling of oxidative phosphoryla-

tion. Although the MPT was originally observed during

necrotic cell death, it also may explain the release of mito-

chondrial pro-apoptotic proteins, such as cytochrome c and

apoptosis-inducing factor. A technique to monitor the MPT

in intact cells has been developed. When cultured hepato-

cytes are incubated at 378C with the acetoxymethyl ~AM!

ester of calcein, the neutral ester crosses the plasma mem-

brane. Cytosolic esterases then hydrolyze calcein-AM to the

free acid form whose ionized carboxyl groups trap and

retain the fluorophore inside the cytosolic compartment.

Calcein displays bright green fluorescence that is indepen-

dent of changes in pH and Ca21. After calcein loading of

hepatocytes at 378C, its diffuse fluorescence in confocal

images shows its distribution within the cytosolic compart-

ment. Within this diffuse fluorescence are many individual

dark voids of about a micron in diameter. To identify these

voids in the calcein fluorescence, cells can be loaded with

tetramethylrhodamine methylester ~TMRM!, a red fluoresc-

ing cationic dye that accumulates electrophoretically in

mitochondria in response to their negative membrane po-

tential. Fluorescence of the two probes is then imaged

simultaneously using LSCM. Virtually every dark round

void in the green calcein image is a TMRM-labeled mito-

chondrion in the red image. Thus, the dark voids in the

calcein images are individual mitochondria. The dark voids

exist for the reason that the mitochondrial inner membrane

is highly impermeable to calcein. When the mitochondrial

membrane degrades, or MPT occurs, individual mitochon-

dria fill up with calcein, resulting in an image with diffuse

calcein fluorescence. This event was accompanied by mito-

chondrial depolarization, as shown by loss of mitochondrial

TMRM fluorescence ~Nieminen et al., 1995!.

Aside from the morphological changes associated with

the nucleus and plasma membrane of cells undergoing

apoptosis, other biochemical changes inside the cell may

have an equally important role in the propagation of apop-

totic signals. One such change involves the release of cyto-

chrome c from mitochondria. Although several mechanisms

for cytochrome c release have been proposed, including

mitochondrial depolarization, outer mitochondrial mem-

brane rupture and opening of permeability transition

pores ~Haunstetter and Izumo, 1998; Heiskanen et al., 1999;

Saraste and Pulkki, 2000!, it remains unclear exactly

how cytochrome c escapes the mitochondria. The function

of cytochrome c outside of the mitochondria is very clear;

cytochrome c interacts with Apaf-1 and caspase-9 to acti-

vate caspase-3 ~Haunstetter and Izumo, 1998!. Therefore,

localization of cytochrome c may provide an early indica-

tion of apoptosis; however, the amount of cytochrome c

necessary in the cytoplasm to trigger apoptosis remains

unknown ~Willingham, 1999!.

The distribution of cytochrome c has been examined in

apoptotic cells using both fluorescence and electron micros-

copy methods ~Heiskanen et al., 1999!. Green fluorescent

proteins ~GFP! can be utilized to detect mitochondrial

events during apoptosis in living cells by LSCM. Recently, a

method to visualize simultaneously cytochrome c release


and mitochondrial depolarization in intact cells has been

developed. Cells transfected with a cytochrome c-GFP fu-

sion construct were loaded with TMRM, a mitochondrial

membrane potential sensitive probe. The green and red

fluorescence of cytochrome c-GFP and TMRM were imaged

by confocal microscopy ~Fig. 7!. The results show that

mitochondrial depolarization accompanied cytochrome c

release during apoptosis. Moreover, individual mitochon-

dria depolarized and released their cytochrome c asynchro-

nously ~Heiskanen et al., 1999!. Besides cytochrome c, other

apoptotic or antiapoptotic proteins, such as Bax, Bid, and

Bcl-2, can be visualized as their fluorescent protein deriva-

tives with confocal microscopy. Using fluorescent-protein-

tagged fusion proteins, one needs to take into consideration

that the fluorescent protein tag may alter the interaction of

the native protein of interest with other proteins. Therefore,

it is advisable to confirm the results by immunocytochemis-

try or immunoprecipitation, if possible.

A fluorescently labeled anticytochrome c antibody was

used to monitor photoirradiation-induced depolarization

of the mitochondrial inner membrane by chloromethyl-X-

rosamine that ultimately resulted in apoptosis ~Minami-

kawa et al., 1999!. These studies showed a dramatic

redistribution of cytochrome c from the mitochondria into

the cytoplasm after photoirradiation. Ultrastructural local-

ization of cytochrome c was examined in tissue sections

Figure 7. Cytochrome c-GFP ~Green Fluorescent Protein! was maintained in the mitochondria before apoptosis and

stained green particles within the cytoplasm. Once it was released during apoptosis, the green particles disappeared and

the cytoplasm became diffusely stained. Cytochrome c-GFP transfected breast cancer cells were loaded with a

mitochondrial membrane potential indicating probe tetramethylrhodamine methyl ester ~TMRM!. Images of green

cytochrome c-GFP fluorescence and red TMRM fluorescence were collected using LSCM. After collecting basal images

~top row!, 1 mM staurosporine was added to induce apoptosis ~bottom row!. In the baseline image, the punctate

fluorescence of green cytochrome c-GFP colocalized with the red TMRM fluorescence ~overlay!. After an 8-hr exposure

to staurosporine ~bottom row!, TMRM and cytochrome c-GFP redistributed from mitochondria to the cytosol as

indicated by a diffuse TMRM and cytochrome c-GFP fluorescence pattern. This indicated mitochondrial depolarization

and cytochrome c release. Bar 5 10 mm.


Table 1. Pros and Cons of Apoptosis Assays

Assay Pros Cons Recommended applications

TEM • Definitive

• Ultrastructural characteristics of

apoptotic cells can be observed

• Time-consuming

• Assays small regions at a time

• Harsh processing conditions

can reduce antigenicity

Sections of regions with lots of

apoptosis or after localization of

cells with thick stained resin

sections

Stained resin sections • Can survey large regions and

sections for TEM

• Detailed tissue analysis possible

• Difficult to maintain antigenicity

for immunohistological or

enzyme assays

Sections with low or high levels

of apoptosis

Caspase-3 activity • Kits available

• Quantitative

• Cannot identify specific cell or

tissue where the apoptosis is

occurring

• Need substantial amounts

of tissue with high levels of

apoptosis

Cells and tissues in culture and

tissue extracts

Acridine Orange • Fast

• Inexpensive

• Fluorescent

• Penetrates tissues

• Toxic

• Mutagenic

• Induces phototoxicity

• Quenches rapidly under standard

conditions

Whole mounts of small embryos

~zebrafish and chick! and tissues

NBS and NR • Fast

• Inexpensive

• Does not penetrate thick tissues

• Lost during preparation for

sectioning

• Hard to visualize in thick or

opaque tissues

Whole mounts of transparent

embryos, organs, tissues

TUNEL • Kits are available

• Well-accepted assay

• Can be used in whole mounts

of thin tissues

• Expensive

• Time-consuming

• Often requires harsh pretreatment

~microwaving or proteinase K!

• False positives possible

Histological sections:

formaldehyde fixed and paraffin

embedded embryos and tissues

or frozen-sectioned and

postfixed samples.

Annexin V • Detects apoptotic cells earlier

than TUNEL

• Allows double and triple

labeling

• Expensive for whole animal

studies

• Variable success with animal

injections

Cell culture and cell sorting,

small embryos

LTR ~Lysotracker Red! • Inexpensive

• Fast

• Penetrates thick tissues

• Stable during fixation and

dehydration

• Resistant to quenching

• Mild staining conditions allow

retention of antigenicity

• Potential false positives for cells

with acidic compartments such

as macrophages and oocytes

• Need to permeabilize to see

through thick tissues in whole

mounts

Whole mounts of tissues, organs,

or embryos and cell culture

PhiPhiLux • Specific to caspase-3/apoptosis

• Assays living cells

• Limited permeability Cell or tissue culture

Mitochondrial assays • Assayed in living cells • Mitochondrial events occur in

apoptosis and necrosis

Cells or tissue culture

Cytochrome C release • Assayed in living and fixed cells Cells or cell culture but has been

used in tissues


from ischemic cardiomyopathic hearts using an anticyto-

chrome c antibody followed by a gold-conjugated second-

ary antibody ~Narula et al., 1999!. In normal heart tissue,

cytochrome c localization was restricted to the mitochon-

dria. In contrast, little cytochrome c was detected in the

mitochondria and substantial staining of cytochrome c was

observed in the Z-bands and intercalated discs of cardiomyo-

pathic hearts.

In summary, a variety of mitochondrial events can be

monitored in living cells over time using multiparameter

LSCM. This technique allows us to assess which mitochon-

drial events are critical for progression of cell injury during

any given pathological condition. The possible limitation of

the technique is laser-light-induced photodamage to living

specimens. However, this can be avoided by attenuation of

the laser power to the minimum possible.

SUMMARY AND CONCLUSION

A wide menu of classic and contemporary techniques cur-

rently available to analyze cell death allows investigators to

select those best suited to specific applications ~Table 1!. No

matter how appropriate and well accepted the assay, it is

recommended that a second assay using a different princi-

ple be used to confirm the detection of apoptosis. More

accurate and precise interpretations of the results of these

assays will become possible as progress continues to be

made in understanding the mechanisms of cell death. The

rapid progress and continued interest in the study of cell

death could spur the development of new protocols or the

modification of old protocols to overcome some of the

current disadvantages with the cell death assays.

ACKNOWLEDGMENTS

The research described in this article has been reviewed and

approved for publication as an EPA document. Approval

does not necessarily signify that the contents reflects the

views and policies of the Agency, nor does mention of trade

names or commercial products constitute endorsement or

recommendation for use. The work was supported in part

by NSF #0074882 ~MW!, NIH HL65314 ~SF!, NIH NS39469

~ALN!, AHA ~SF, MW, FR!.

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