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In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.
A Negoescu, P Lorimier, F Labat-Moleur, C Drouet, C Robert, C Guillermet, C Brambilla and E Brambilla J Histochem Cytochem 1996 44: 959 DOI: 10.1177/44.9.8773561 The online version of this article can be found at: http://jhc.sagepub.com/content/44/9/959

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The Journal of Histochemistry and Cytochemistry Copyright 0 1996 by The Histochemical Society, Inc

Vol. 44, No. 9. pp. 919-968. 1996 Printed in U5.A.

Original Article

In Situ Apoptotic Cell Labeling by the TUNEL Method: Improvement and Evaluation on Cell Preparations
ADRIEN NEGOESCU,' PHILIPPE LORIMIER, FRANCOISE LABAT-MOLEUR, CHRISTIAN DROUET, CATHERINE ROBERT, CHRISTIANE GUILLERMET, CHRISTIAN BRAMBILLA, and ELISABETH BRAMBILLA
Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonnrot (AN,PL,CR,CB,EB),Laboratorre de Pathologie Celhlaire, CHRU (FLM,CG,EB),and INSERM Unrti 238, CEA DBMSIICH Centre &Etudes Nucliarres (CO) Grenoble, France
Received for publication November 15, 1995 and in revised form March 22, 1996, accepted March 26, 1996 (5A3835)

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling)is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivityby optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three pmtmls on CEM-C7 cells, a model of glucocorticoid-indud apoptosis. Samples were submitted to six modalities of fuation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter

two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fmtion, miaowavepretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existenceof a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state. (JhlisfochemCyfochem44:959-%8,
1%)
KEY WORDS: " E L ;

Detergent; Enzymatic digestion; Microwaves; Double labeling; Chemiluminescence; Preapoptosis.

Introduction
For many years, and mainly in the field of oncology, interest was focused on cell division. In the wake of the pioneering work of Kerr et al. (1972),the importance of cell loss through apoptosis (active cell death, ACD) and necrosis (passive cell death) has been increasingly recognized, and the focus is now on the balance between cell proliferation and cell elimination, and the search for a simple and efficient means of specific identification and quantification of the two phenomena. The identification of apoptotic cells relies chiefly on their morphological pattern, involving chromatin condensation and nuclear and cytoplasmic fragmentation into apoptotic bodies (Kerr et al., 1972).The apoptotic nucleus can also condense without splitting (Staunton and Gaffney, 1995),and it is now believed that most of the so-called pyknotic nuclei correspond to the nonfragmented

Correspondence ta A. Negoescu, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Faculti de Mtdecine, Domaine de la Merci. 38706 La Tronche Cedex, France.

'

apoptotic nucleus (Majno and Joris, 1995;Kressel and Groscurth, 1994;Wyllie, 1980;Wyllie et al., 1980). Apoptotic cells are not easily distinguishable by light microscopy from other elements with condensed chromatin, such as mitotic cells in telophase (Staunton and Gaffney, 1995;Migheli et al., 1994,1995; Wijsman et al., 1993; Gavrieli et al., 1992).Moreover, nuclear morphology strongly depends on sample fixation, which influences the identification and interpretation of the morphological aspects of apoptotic cells. Furthermore, owing to the rapidity of the phenomenon (2-5min for chromatin condensation and cell fragmentation, 3 hr for apoptotic body phagocytosis and digestion) (Tenniswood et al., 1992;Bursch et al., 1990a,b),strong ACD-induced tissue decrease is often based on less than 5% of the cell population of histologically visible apoptotic cells and bodies (Bursch et al., 1985,1990). Therefore, an increasing number of investigatorsfavor in situ labeling of apoptotic cells, which allows both high sensitivity and precise identification of the cell population involved. In this approach, specificity is secured by simultaneous evaluation of apoptotic cell morphology. The technique relies on the use of exogenous enzymes, such as DNA polymerase I (Gold et al., 1993;Wijsman et al., 1993;Fehsel et al., 1991;Iseki, 1986;Iseki and Mori, 1985) and terminal deox959

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IN SITU APOFTUIIC CELL LABELING

BY TUNEL

ynucleotidyl transferase (TdT) (Gold et al., 1994; Migheli et al., 1994; Gavrieli et al., 1992; Modak and Bollum, 1970,1972), to incorporate labeled nucleotides into the 3-hydroxyl(3OH) recessed termini of DNA breaks. In general, and without regard to the incorporating enzyme, such techniques are termed in situ end-labeling (ISEL) and, more specifically, in situ nick translation (ISNT) when they involve the use of DNA polymerase I or its Klenow fragment, or TdT-mediated dUTP nick end-labeling (TUNEL) when they employ terminal transferase. TUNEL is considered more sensitive and specific than ISNT (Gold et al., 1994; Migheli et al., 1994; Gorczyca et al., 1993), and TUNEL has been chosen for the two main commercially available in situ apoptosis detection kits produced by Oncor (Gaithersburg, MD), ApopTag and ApopTag Plus, and by Boehringer (Mannheim, Germany). The accessibility to DNA breaks for enzymatic reactions is reduced by the nuclear protein environment (Kerrigan et al., 1987). This main drawback for TUNEL is worsened by cell and tissue fixation (Gold et al., 1994). Therefore, chemical pretreatments of the biological samples have been devised to improve TUNEL sensitivity. At present, on cultured cells TUNEL is performed after paraformaldehyde (Gold et al., 1994)or formaldehyde (Gavrieli et al., 1992) fixation, sometimes followed by ethanol postfixation (Gorczyca et al., 1993). When pretreatments are used, they consist of incubating cells with a detergent, most frequently Triton (Desjardins and MacManus, 1995; Kressel and Groscurth, 1994; Sgonc et al., 1994) which, in our hands, exhibited very limited efficiency. Crosslinking of proteins due to fixation is considered to be the cause of reduced accessibility to nuclear DNA for enzymatic reactions (Gold et al., 1994). and this has prompted the pretreatment of tissue s a m ples with proteolytic enzymes, mainly proteinase K, to enhance TUNELsensitivity (Gold et al., 1994; Migheli et al., 1994; Gavrieli et al., 1992). an approach not yet applied to cultured cells. Because one of the effects of the widely used microwave antigen retrieval method (Catoretti et al., 1993; Shi et al., 1991) is peptide bond cleavage (Wu et al., 1992), we assumed that this approach, together with proteinases, might prove efficient for TUNEL applied to cultured cells. Two articles (Lucassen et al., 1995; Strater et al., 1995). published while this manuscript was in final preparation investigated advantages and drawbacks related to TUNEL staining of formaldehyde-fixed, paraffin-embedded, microwave-pretreatedtissue sections. The present study aimed at (a) improving the sensitivity of TUNEL, while retaining specificity, by testing pretreatments not previously performed on cells, i.e., microwave irradiation and the widely tissue used proteinase K and (b) extending TUNEL applicability to a large panel of fixatives that may be imposed by common practice or by association with other techniques, such a s TUNEL-protein or TUNEL-nucleic acid in situ hybridization double labelings. Our results showed that, with adequate pretreatment, sensitive and specific TUNEL can be obtained on cultured cells fixed not only with paraformaldehyde or formaldehyde but also with B5, Bouins liquid, methanol, and acetone. Whatever the fixation, the microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity. The existence of TUNEL-stained cells that lacked apoptotic morphology contributes to the current debate about the notion of preapoptotic state (Migheli et al., 1994,1995; Gorczyca et al., 1993,1994; Gavrieli et al., 1992).

Materials and Methods

Cell Cultures
TUNEL improvement experiments were performed on cultures of CEM-C7 cells, a cloned human leukemic T-cell line derived from childhood acute lymphoblastic leukemia (Foley er al., 1965). which is characterized by its significant apoptotic response to glucocorticoid incubation (Alnemri and Litwack. 1990). CEM-C7 cells were grown in suspension in 0.0523% (wiv) N-acetyl-~-alanyl-~-glutamine-enriched RPMI 1640 medium (Biochrom; Berlin, Germany) containing 10% (viv) fetal calf serum (Gibco; Gaithersburg, MD), 1% (viv) nonessential amino acids (Gibco). and 1 mM sodium pyruvate (Sigma-Aldrich; St Quentin Fallavier, France) at 37C under 5 % COz in a humidified atmosphere. To trigger apoptosis. CEM-C7 cells were incubated for 3 hr with 13 pM dexamethasone (Soludecadron; Merck. Darmstadt, Germany), conditions providing the optimum apoptotic cell yield. Cells were washed with 0.1 M PBS, pH 7.4. at 4C and then were used for TUNEL or DNA analysis by electrophoresis. TLJNEL-p53 double labeling was performed on 4% paraformaldehydefixed cultures of H 322 cells, a cloned human non-small-cell lung cancer (NSCLC) line, which accumulate mutated p53.

Fixation
Six fixatives frequently used in pathology and cytology were tested: 4% (wiv) paraformaldehyde (PFA) in PBS; 4% (viv) formaldehyde (formalin) in PBS; B5 [ 8 % (wiv) mercury chloride in 4% formaldehyde]; Bouins liquid [2.5% (wiv) copper acetate, 4% (wiv) picric acid in 3.5% (viv) formaldehyde, 1.5% (viv) acetic acid in distilled water]; methanol (at -20-C); and acetone (at -20C). CEM-C7 cells were fixed for 10 min at 4C, washed with PBS at 4C. then cytospun on aminopropyl-triethoxysilane(APES)-treated glass slides and allowed to dry at room temperature (RT).

TUNEL
Three modalities of TUNEL were tested throughout the study: an original laboratory protocol and the kits from Boehringer and from Oncor (ApopEg). The laboratory protocol was adapted from Billig et al. (1993), Gavrieli et al. (1992), Gold et al. (1994), Gorczyka et al. (1993), and Migheli et al. (1994,1995).Nonspecific labeling was prevented by preincubating samples with 3% bovine serum albumin (BSA) 20% normal bovine serum (Jackson; West Grove. PA) in PBS for 30 min at RT. The specimens were then incubated for 1 hr at 37C in a moist chamber with the TUNEL mix, which consisted of 0.13 5 Uipl calf thymus terminal deoxynucleotidyl transferase (TdT), 0.0044 nmolipl digoxigenin-~l-2-deoxyuridine-5-triphosphate (DIG-dUTP), and 1 mM cobalt chloride in 1 x reaction buffer in distilled water. All reagents in the TUNEL mix were obtained from Boehringer. After washing (four PBS baths of 5 min each at RT), the specimens were resaturated (30 min at RT) in 3% BSA and 20% normal sheep serum (Jackson) in 1% (w/v) blocking reagent (Boehringer) in 0.1 M Tris-buffered saline (TBS). They were then treated for 1 hr at RT with a 1:120 (1.25 peroxidase Ulml) dilution of peroxidase-labeled anti-digoxigenin sheep Fab fragment (Boehringer), followed by washing and 0.05% 3,3-diaminobenzidinetetrahydrochloride (DAB) color reaction. The antibody was diluted in PBS containing 1% BSA and 0.01% sodium (ethylmercurithio)-2 benzoate (Prolabo; Paris, France). The Boehringer kit was used according to the manufacturers instructions, with the addition of two normal serum saturations as mentioned above, since this kit uses calf thymus TdT, fluorescein-dUTP, and a peroxidaselabeled anti-fluorescein sheep Fab fragment. A 1:2 dilution of the antibody gave optimal specific signa1:background ratio

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The ApopTag kit was used strictly according to the manufacturer's instructions. This kit uses DIG-dUTP and a peroxidase-labeled anti-DIG antibody. However, since neither the origin of the TdT nor that of the antibody are mentioned in the documents provided with the kit. we introduced no additional saturations. Counterstaining was performed with Harris' hematoxylin. Slides were dehvdrated. mounted in Merckoglas (Merck). observed with a BH2 Olympus microscope, and photographed on 100 Gold films (Kodak-Pathe. Paris, France).

2027
564

Pretreatments
TUNEL was performed without Pretreatment and with each of the following three pretreatments. Preliminary tests allowed the operating conditions to be adjusted for optimal results. Once established, each optimal method was reproduced at least in triplicate and quantification was performed on a slide representative of each working condition. (Sigma-Aldrich) was used at 0 . 1 1 (vlv) for 2 Detergent. Triton X-100 min on ice, followed by washing of the slides twice in PBS at RT. This is the most widely-used procedure (Desjardins and MacManus. 1995; Kressel and Groscurth, 1994; Sgonc et al.. 1994) and its use is also recommended in the Boehringer kit. Proteolytic Enzyme. Proteinase K (Boehringer) was employed at 25 pglml in PBS for 15 min at RT. Slides were then rinsed twice in PBS. Preliminary tests consisted of varying concentration [2. 10. 25. and 40 Bglml. according to Migheli et al. (1994)] and incubation time (5, 10. and 15 min). Microwave Irradiation. We employed an AEG Micromat 120 domestic microwave oven operating at a frequency of 2.45 GHz with nine power level settings. the highest power being 850 W. Best results were obtained with Setting 4 (370 W). The slides were transferred to a plastic jar containing 200 ml of 0.01 M citrate buffer, pH 6, and irradiated for 5 min. resulting in elevation of the temperature from RT to 86C. Immediately after irradiation. the jar was filled with an extra 80 ml of distilled water at RT to cool the retrieval solution; then the slides were quickly immersed in PBS at RT (rapid cooling). This type of buffer has been recommended for microwave enhancement of TUNEL (Lucassen et al., 1995; Strater e t al., 1995) and of mRNA in situ hybridization (Sibony et al.. 1995) in tissue sections. Preliminary tests included power variation from Setting 2 (188 W, 54'C) to Setting 8 (752 W, 98'C boiling) for one or two 5-min periods; we also compared classical 15-min progressive cooling in the citrate buffer (Shi et al.. 1991) vs rapid cooling. Microwave Irradiation Followed by Proteinase K Digestion. The protocol was that defined as optimum by Strater et al. (1935), i.e.. the slides were immersed in 50 ml of citrate buffer and irradiated for 1 min at 750 W (86'C). rapidly cooled. then incubated for I5 min at RT in 20 Vglml proteinase K. The absence of peroxidase in our biological samples (data not shown) made inhibition unnecessary. This was an advantage. because H202 pretreatment has been reported to weaken TdTactiviry (Migheli et al.. 1995) and to induce DNA breaks (Wijsman et al., 1993).

125

DEXAMETHASONE
Figure 1. Agarose gel electrophoresis of apoptotic DNA of dexamethasonetreated CEM-C7 cells (Lane 1) compared to control DNA (Lane 2).

The specificity was controlled by comparing TUNEL labeling with cellular morphology (see below). To assess possible DNA fragmentation induced by microwave pretrearmenr, we used a very sensitive means of detection, the chemiluminescent luminol (ECL: Amersham, Amersham. UK), a substrare of peroxidase. as previously described (Lorimier et al.. 1993). The luminescent signal was picked up by a photon collecting system consisting of an Olympus BH2 microscope equipped with a photon intensifier connected to a CCD camera (IHCD 2400-47; Hamamarsu. Hamamatsu City. Japan) and was visualized on a color video monitor (Sony; Tokyo,Japan). Cells were first located in transmitted light on the screen to facilitate morphological identification. The images were obtained by photographing the screen. TdT-free negative controls provided the threshold level for chemiluminescent detection.

TUNEL-Protein Doubk Labeling

The TUNEL-p53 double labeling was performed on H322 cells in a sequential manner. After TUNELadapted microwave pretreatment (370 w ) , p53 immunostaining, the first step. was effected by a series of incubations: overnight at 4'C with mouse monoclonal IgG anti-human p53 clone DO7 (1:50; Dako, Glosrrup. Denmark), followed by biotinylated donkey antimouse F(ab)* (1400: Jackson) for 1 hr at RT. The alkaline phosphatase substrate was Fast Blue (FB; Sigma, St Louis. MO). After the FB color reaction was completed and the slides washed. TUNEL was performed with the Boehringer kit. No counterstaining was performed. The slides were mounted in Aquamount (BDH Gurr; Poole. UK).

Controfs
A negative control for apoptosis induction was obtained by eliminating dexamethasone incubation of the CEM-C7 cells and was confirmed by analysis of the characteristic apoptotic DNA ladder obtained in agarose gel electrophoresis according to the method of Banda et al. (1992) (Figure 1). To provide a negative control for the TUNEL, we omitted TdT from the labeling mix.

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IN SITU APOPTOTIC CELL LABELING BY TUNEL

Cell Counting and Statistical Analysij

CEM-C7 cells were assessed for morphology typical of apoptosis and staining according to Gold et al. (1994). Briefly (Hollwoodand Macartney, 1991; Kerr et al., 1972), the followingchangeswere considered to represent apoptosis and were counted as such, at x400 magnification: (a) marked condensation of chromatin and cytoplasm (apoptotic cells); (b) cytoplasmic fragments with or without condensed chromatin (apoptotic bodies);and (c) intra- and extracellularChromatin fragments (micronuclei). Values given in Table 1 represent percentages from at least 1000 counted apoptotic and nonapoptotic cells. Statisticalanalyses were performed using the exact Fisher test. Differences were considered as significant when p < 0.05.

apoptotic bodies. Fixative-inducedmorphological variations did not perturb labeling interpretation.

E f f t of Pretreatments
Without pretreatment, TUNEL had poor sensitivity, with 3-17% of cells with apoptotic morphology labeled (Figure 3a) for five of six fixatives used. Acetone provided the best sensitivity without pretreatment, allowing 5 5 % of morphologically apoptotic cells lacking apoptotic morphology to be marked. In these conditions, TUNEL specificity was high, with a maximum of only 5 % labeled cells lacking apoptotic morphology, including some mitoses, the labeling of which was occasional but generally intense. The most frequently labeled mitoses were observed with methanol fixation, and they were exceptional in B5-fixed C E M O cells. No necrotic CEM-C7 cells could be identified. Whatever the fixative, TUNEL sensitivity was virtually always improved by pretreatments (Table 1). The best sensitivity enhancer was microwave irradiation (Figure 3c), with a four- to 30-fold increase in the percentage of labeled cells with apoptotic morphology for all fixatives except acetone, for which the percentage was almost doubled. Notably, detergent (Triton) pretreatment had a low efficiency,with less than one third of the morphologically apoptotic cells being marked (Figure 3b). Proteinase K was superior to Triton, with percentages reaching 50% for BS-fixed cells, and even more than 80% for methanol and acetone fixations. Combined microwave-proteinase K pretreatment never proved more efficient than microwaves alone. For four of six fixatives(PFA, formaldehyde, B5, Bouin), pretreatments did not induce important losses in specificity, with a limited proportion of mitoses and normally shaped cells being marked (Figures 4a-4c). In contrast, a high percentage of labeled morphologically normal cells (including mitoses) were observed in cells fuced with methanol or acetone after treatment with proteinase K or microwaves, either separately or in combination. However, this increase was three times smaller in cells pretreated exclusively with microwaves (Table 1).

Results
The results are summarized in Table 1.

Efect of TUNEL Protocol

The three modalities of TUNEL we have tested (laboratory protocol, ApopTag kit, and Ebehringer kit) were not statisticallydifferent regarding sensitivity and specificity. In this context, the highest percentages of TUNELpositive, morphologically apoptotic cells were scored using the Boehringer kit. Moreover, this kit gave the best signakbackground ratio while being the easiest to use of the three protocols.

Fixzztiues and Cell and Labeling Morphology

Nuclear morphology and chromatin appearance were influenced by the type of fixation, the highest resolution being provided by Bouinsliquid and the lowest by methanol; nevertheless, the criteria of Kerr et al. (1972) for apoptotic cell identification by light microscopy could be observed clearly whatever the fixative (Figures 2 and 4). TUNEL coloration of nuclei and micronuclei was generally heterogeneous, with reinforcement at the nuclear border. Occasionally the brown color expanded over the cytoplasm, mainly in small

Table 1. Percent of TUNEL-positive CEM C-7 cells with apoptotic ana nonapoptotic morphology a,b
Pretreatment Fixative None
Apo

Triton Nonapo
0
1

Proteinase K
Apo 26 41* 50* 17 82**

Microwaves Apo
89 89 87 84 93 92

Microwaves and Proteinase K

Apo

Apo

Nonapo
0 1 2

Nonapo

Nonapo

Nonapo

Paraformaldehyde Formaldehyde B5
Bouins liquid Methanol Acetone

3
14* 15* 17*

5
0 5

6
55**

13 14 30* 17 15 55**

3
2
8 41*

5
0 15*

3 3 2 8
15*

66*
71*

3
3

66*
17 82** 92**

2 5
41* 92**

92**

92**

34**

For each fixative. preliminary counts identified statistically equivalent working conditions and TUNEL modalities (laboratory, ApopTag kit, Boehringer kit), which were pooled and a global percentage is given in the table. The three modalities of TUNEL (laboratory, ApopTag kit, Boehringer kit) did not give significantly different results. Therefore. the apparent differences within the table horizontal lines (between pretreatments for the same fuative) are all significant (p<O.OS). When percentages in each vertical column were compared (between fixatives for the same pretreatment), significant differences were found (p<0.05) between values afTected with different numbers (none to two) of asterisks: e.g.. in the first column, 55** is significantly larger than 17*. itself significantly greater than 6; but 1S* is not significantly larger than 14*. Apo, cells with apoptotic morphology; Nonapo, cells with nonapoptotic (normal) morphology. Values given in the table represent percentages of at least 1000 Apo or Nonapo counted cells.

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NEGOESCU, LORIMIER, LABAT-MOLEUR, DROUET, ROBERT, GUILLERMET. BRAMBILLA. BRAMBILLA

%3

Figure 2. TUNEL detection of apoptotic cells on variously fixed CEM-C7 cells: 4% paraformaldehyde (PFA, a); 4% formalde) ; 85 (c); Bouin's liquid (d); hyde (formalin, b methanol (e); acetone (1). Arrows point to cells exhibiting typical apoptotic morphology. Microwave pretreatment. TUNEL performed with the Boehringer kit. Original magnification x 132.Bars = 5 pm. Figure3 . TUNEL enhancement by pretreatments. (a) Without any pretreatment, regardless of the fixative. TUNEL sensitivity is insufficient: manyCEMC7cells with apoptotic morphdogy are unlabeledor weakly labeled (arrowhead). (b) Detergent induced very limited sensitivity improvement. with still many unlabeledepoptotic cells (anowhead). (c)The best pretreatmentcondition with reference to apoptotic morphology proved to be microwaves. Fixation by Bouids liquid. TUNEL performed with the Boehringer kit. Original magnification x 132. Bars = 5 pm.

Proteinase K at 2 pg/ml did not improve TUNEL sensitivity. regardless of the incubation time. Results were variable at 10 pglml for all three incubation times (5, 10, and 15 min) and at 25 pglml for the 5-min and 10-min incubations. At 40 pglml, almost all nuclei were marked by TUNEL. Analogously, microwave pretreatments at less than 370 W for 5 min had low dficiency in improving TUNEL sensitivity, and at greater energy input (higher power settings or repetitions of the 5-min irradiation period) most nuclei became TUNELpositive. Rapid cooling improved the signal:background ratio. With regard to cell morphology, the impact of pretreatments was negligible except for proteinase K-treated cells fixed with ace-

tone or methanol, which showed some ruptured cytoplasmic and nuclear membranes.

Controls
Apoptosis Controls. To follow ACD induction by dexamethasone in CEM-C7 cells, we traced apoptosis via overall DNA fragmentation as detected in agarose gel electrophoresis. The negative controls for ACD were CEM-C7 cells cultured without dexamethasone. This approach showed DNA fragmentation, as assayed by a nucleosomal ladder, only after dexamethasone treatment (Figure 1). T h i swas in agreement with the microscopic quantification,which indicated that 25 i 5% of dexamethasone-treated CEM-C7 cells

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! % 4

I N SITU APOPTOTIC CELL LABELING BY TUNEL

Figure 4. TUNEL specificity. The fragmented nucleus of advanced apoptosis (a, open arrow) isstronglylabeled,asis thecrescentlike chromatin condensation @, closed arrow). Rare nuclei with normal appearance are stained (a, closed arrow). Mitoses are either unstained (b. open arrow) or strongly labeled(c,dosed a " ) Microwares . do not inducediffuseDNA lesions, as shown by the f the sensiclear-cut all-or-nothing patterno tive luminescence detection: the apoptotic body shown in transmitted ligM (d, a r m head) is heavily luminescent (e), whereas the surrounding normal nucleiare all negative. PFA-fixed. microwavepretreatedCEMC7 cells. TUNEL performed with the laboratory protocol. Original magnification x 260. B a n = 5 pm. Figure 5. TUNEL (DAB, brown)-p53 (Fast Blue) double labeling on H-322cells. Note the brown nuclei at the beginningof apoptotic shrinkage (short arrow) or with crescent-like labeling (arrowhead). Mixed bluebrown color indicates the presenceof p53 in a nucleus initiating active cell death (long arrow). PFAfixed, micmavepre treated cells. TUNEL performedwith the l a b oratory protocol. Original magnification x 132. Bar = 5 em.

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965

showed apoptotic morphology. This proportion fell to 1 1% in the absence of dexamethasone ( p < O.OOOl), and these apoptotic cells were intensely labeled by TUNEL. Interestingly, labeled cells with a nonapoptotic aspect were few in dexamethasone-freeCEMC7 cell cultures, even after microwave pretreatment (0-1% for PFA, formalin, B5, and Bouin, 5% for methanol and acetone, compared with 2-34% in the presence of dexamethasone;p < 0.05).

TUNEL Control. Lack of TdT in the TUNEL mix completely abolished labeling under all working conditions.
Microwave E f k t Control Through ChemilumineSQnce. Because microwaves at high energy input induced staining of all CEM-C7 cell nuclei (see above), we feared that this pretreatment might alter DNA integrity, creating TUNEL false-positive nuclei. Under our working conditions (370 W), DAB coloration was restricted to cells bearing apoptotic morphology and to a small minority of the rest. To verify that the nuclear staining was not simply hidden by insufficient sensitivity of chromogenic detection, we submitted our cell preparations to the chemiluminescent system, 100 times more sensitive than DAB, as previously described (Negoescu et al., 1994; Lorimier et al., 1993). Whatever the fixative, luminescent detection showed a majority of entirely negative nuclei (Figures4d and 4e).

We investigated TUNEL adaptability to various sample conditions that were likely to be imposed by current laboratory practices or immunochemical procedures aiming at TUNEL-protein double labeling. Our results showed that by means of pretreatments, among which microwaves proved the best, TUNEL sensitivity can be extended over a range offixativeswithout notable impairment of the specificity, and that compromises can be found between TUNEL and immunochemical labeling requirements. T h i s was achieved with each of the three modalities of TUNEL we tested (an original laboratory protocol, ApopEg kit, Boehringer kit). However, we admit a slight advantage for the Boehringer kit in terms of sensitivity, signakbackground ratio, and convenience of use.

Pretreatments and TUNEL Sensitivity

Sample pretreatment before TUNEL proved indispensable. In the absence of any pretreatment, even with the especially favorable acetone fixation, TUNEL sensitivity reached only 55 %; with the other fixatives it was very low, with less than 20% of morphologically apoptotic cells being marked. The choice of pretreatment was of basic importance. Indeed, detergent, the only type of pretreatment reported for cultured cells (Desjardinsand MacManus, 1995; Gold et al., 1994; Kressel and Groscurth, 1994; Sgonc et al., 1994), had little or no effect in improving TLJNEL sensitivity on C E M G cytospots (Table 1). The gain in sensitivity was about twofold for microwaves compared to proteinase K, itself globally twice as effective as detergent. Proteinase K-microwave combination, as optimized for tissue sections by Strater et al. (1995), gave intermediate results between each of the two pretreatments used singly. Widely used for epitope retrieval, microwaves may also be useful for TUNEL-immunochemistry double labelings. .For example, working conditions could be readily adapted for the double labeling of 3-322 cells presented in Figure 5 , since PFA fixation and microwave pretreatment are appropriate for both optimal TUNEL on cell cultures and p53 immunohistochemistry (Tenaud et al., 1994). Even with the pretreatment producing the highest sensitivity, i.e., microwaves used in the limits of controlled specificity, about 10-20% of nuclei whose morphology was clearly apoptotic remained unstained. On paraformaldehyde-fixed cells we obtained almost 90% of labeled morphologically apoptotic cells after microwaves. Gold et al. (1994), who performed the only quantitative comparison between TUNEL and apoptotic morphology, published results of nearly 100% of TUNEL-stained apoptotic nuclei on X-rayirradiated thymocytes after permeabilization by a detergent (Nonidet P-40). Using a detergent (Triton X-100). we reached only about 10%labeled cells among those with the apoptotic morphology. The tendency of X-rays themselves to fragment DNA (Elia et al., 1991), even when the proper intensity ranges to induce apoptosis (Umanski, 1991) are respected, may account for a higher number of DNA breaks per cell, resulting in greater TUNEL sensitivity. The exact mechanism underlying the increase of TUNEL sensitivity as a result of microwave pretreatment remains largely obscure. Fixation-inducedcrosslinking (Mason and O'Leary, 1991)or precipitation in the protein environment of DNA breaks (Kerrigan et al., 1987), as well as DNA-DNA crosslinkage (Fox et al., 1985), presumably reduces the accessibility of 3'OH recessed ends for TdT, accounting for the beneficial effect of both proteolytic (Gold et

TUNEL-Protein DoubZe LabeZing

On H-322 cells, TUNELstained condensed nuclei of apoptotic appearance, micronuclei, and some normally shaped nuclei. Some nuclei of H-322 cells were labeled for both TUNEL and p53 (Figure 5).

Discussion
Active cell death (ACD) is best characterized by a series of morphological changes (Kerr et al., 1972). The second cardinal feature of apoptotic cells is DNA fragmentation, although it does not always evolve according to the scheme ofinteroligonucleosomalbreakage that results in the DNA ladder (Desjardins and MacManus, 1995; Sun et al., 1994; Walker et al., 1993; Brown et al., 1992; Wyllie et al., 1980,1984).Taken separately,each of these two characteristics of apoptotic cells presents serious drawbacks in identification and quantification of ACD. In situ end-labeling (ISEL), by coupling the two approaches, morphology and DNA fragmentation, into a unique assay has therefore rapidly become the method of choice in apoptosis investigation. This generated critical studies of the two ISEL variants, with TdT-mediated dUTP nick end-labeling (TUNEL) being generally preferred to DNA polymerase I-based in situ nick translation (ISNT) in terms of sensitivity and specificity (Gold et al., 1994; Migheli et al., 1994; Ansari et al., 1993;Gorczyca et al., 1993). Greater TUNEL efficiency has been explained by the assumption that double-strand DNA breaks occur at a higher frequency than single-strand breaks in apoptotic nuclei. TdT tailing of both single- and double-strand breaks would therefore perform better than DNA polymerase I, which requires one intact DNA strand (Gold et al., 1994). TUNEL has been almost exclusively applied to cells fixed with paraformaldehyde or formaldehyde, the first proposed fixatives.

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al., 1994) and microwave pretreatments in the TUNEL protocol. Microwaves cleave peptide bonds in a more selective fashion than simple heating (Wu et al., 1992), probably by increasing molecular agitation (Catoretti et al., 1993). Indeed, we observed (data not shown) that reducing heat input by rapidly cooling the samples after irradiation, instead of the 15 min of progressive cooling classically recommended (Shi et al., 1991), distinctly increased the TUNEL signallbackground ratio. On the other hand, when the primary interest is the protein expression rather than the quantity of the apoptotic cells, a limited loss of TUNEL sensitivity does not preclude correct interpretation of results and therefore can be accepted as the price of the compromise between TUNEL and immunohistochemistry requirements. In this regard, acetone fixation without pretreatment, despite its approximately 50% sensitivity, may be of interest in situations when pretreatments are precluded, such as cells grown on organic gels.

Pretreatments and TUNEL Speczficity

Concerning TUNEL specificity, and seemingly in agreement with all other investigatorsresults, we approve Ansaris formula that ISEL technique is selective (rather than specific) for apoptotic nuclei, since these contain a far greater degree of DNA fragmentation. (Ansari et al., 1993) The notion of degree appears central, because DNA fragmentation is associated with various biological and artifactual situations, such as necrosis (Kerr and Harmon, 1991), DNA recombination, replication, repair or compaction-relaxation during mitosis (Wolffe, 1995; Eastman and Barry, 1992; Eli, et al., 1991), sample fixation, paraffin embedding and cutting (Sasano, 1995; Ansari et al., 1993; Wijsman et al., 1993), and detergent and proteinase K pretreatments (Migheli et al., 1994; Szabd et al., 1990). In this respect, the microwave effect remains questionable. The goal of TUNEL procedures can be understood as an attempt to find a threshold at which only cells with a high degree of DNA fragmentation are labeled. In our preliminary experiments we observed, as did Migheli et al. (1994), that excessive proteolytic pretreatment induced the TUNEL staining of almost all nuclei on cell preparations and tissue sections. Like Lucassen et al. (1995), and in contrast to Strater et al. (1995), we observed that highly energetic microwave pretreatment resulted in general nuclear coloration. We considered as optimal those conditions in which a majority of apoptotic nuclei, and the smallest percentage of the remaining nuclei, were marked by TUNEL. Under such conditions we could not avoid strong staining of a proportion of mitoses and of some normally shaped cells. Mitosis labeling, already reported by Migheli et al. (1994). may be linked to topoisomerase 11-induced double-strand breaks during DNA compaction into chromosomes and chromosome separation at the end of division (Wolffe, 1995; Eastman and Barry, 1992; Elia et al., 1991). At variance with mitotic and necrotic cells whose morphology is distinct from apoptosis, the status of normally shaped, intensely TUNEL-positive cells is a matter of debate: are they in a preapoptotic stage, as suggested by Gavrieli et al. (1992). Gorczyca et al. (1994), and Migheli et al. (1994, 1995)?These cells would correspond to the stage at which high molecular weight DNA fragments can be detected before changes in cell morphology occur (Walker et al., 1993). This interpretation is supported by the very low number

of such elements in CEM-C7 cell cultures grown free of dexamethasone, the inducer of ACD. It is also sustained by the recent detailed investigation of Desjardins and MacManus (1995), who determined distinct morphological and biochemical stages in teniposide-induced apoptosis of colon adenocarcinoma HT-29 cells. They proposed a four-stage scheme ranging from intact control cells to floating apoptotic bodies. DNA begins to break into large (loo-5oo KBP) fragments at Stage 2, whereas complete apoptotic morphology and the DNA ladder exist only at Stage 4. TUNEL becomes positive only at Stage 4, a result explained as follows: It is likely that the sensitivity of our [TUNEL] technique was such that a small number of large DNA fragments could not be detected. This conclusion is in agreement with our low positive results using, as they did, a Triton pretreatment. The improvement in sensitivity obtained by proteolytic digestion or microwave irradiation may be attributed to detection of lowerdegree DNA fragmentation, natural or artifactual. The first situation to be evoked is that of DNA breaks induced during DNA repair. However, such a mechanism has already been rejected because virtually every cell has such breaks (around 100 KBP per cell) and labeling should therefore uniformly affect all cells and not only a subset (Migheli et al., 1995; Mundle and Raza, 1995). The same reasoning can be extended to artifactual DNA breaks (Sasano, 1995; Ansari et al., 1993; Wijsman et al., 1993). As a matter of fact, histological specimens submitted to long-term fixation, embedding, and cutting actually displayed more normal labeled nuclei than CEMC7 cytospots that were not submitted to these manipulations (data not shown). Detergents (Nonidet P-40) and proteinase K are reported to break DNA (Migheli et al.. 1994; Szabd et al., 1990). Accordingly, they are used below certain limits of concentration, temperature, and exposure times. This also appears to be true for microwaves: they are known as an efficient enhancer of both DNA (Kok and Boon, 1992; van den Brink et al., 1990) and RNA (Sibony et al., 1995) in situ hybridization, and this would not be the case if they caused a loss of target sequences due to nucleic acid strand breaks. However, highly energetic microwave pretreatment probably fragmented DNA, because most CEM-C7 nuclei were marked by TUNEL. This was not the result with a lower energy input, which only allowed staining of the morphologically apoptotic cells and a minority of the rest, a clear-cut all-or-nothingpattern confirmed by the very sensitive chemiluminescence detection (Lorimier et al., 1993; Negoescu et al., 1994). The above considerations imply the necessity to routinely apply a number of critical criteria to obtain a sensitive and specific evaluation of apoptosis by means of TUNEL. First, if comparative quantification of apoptosis is desired, one should avoid using Samples fixed according to various modalities and should standardize pretreatment. Second, labeling should be accepted as specific only if it is strong, as compared to general background, and concerns isolated cells lacking mitotic or necrotic characters. Third, labeled cells devoid of apoptotic morphology should be accepted as preapoptotic provided that they are associated with strongly stained elements with classical apoptotic features on the same cell or tissue preparation. Finally, automatic methods such as flow or morphometric counts, should be supported and assessed by reference to morphological evaluations.

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Acknowledgments
W e thank Dr Ruth Griffin-Shea, who kindly revised the English version ofthis manuscri& We are indebted to Catherine Pipin andJean-Michel

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Lasserre for the photography.

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