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TECHNICAL

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Simultaneous detetmlnation of cell mLrfaceantigens and apoptosis Apoptosis is important in a variety of cellular events. The process is morphologically characterized by membrane blebbing, a decrease in cell volume, increased cell density, compaction of cell organelles and chromatin condensation. The most striking feature is cleavage of the cell's DNA at intervals of-200 bp by endogenous endonuclease(s): this produces a typical 'ladder' of DNA fragments on electrophoresisl. At present, apoptosis can be detected by genomic DNA analysis, which cannot determine the state of individual cells, by staining DNA with a variety of fluorochromes, or by a combination of sorting immunofluorescence-stained cells and DNA analysis, which is very time consuming and requires relatively large amounts of cells (reviewed in Ref. 2). Gavrieli et al.~ recently reported a new method for detecting apoptosis in tissue sections, terminal deoxynucleotidyl transferase-(TdT) mediated dU'IP-biotin nick end labeling (TUNED of DNA fragmentation sites in nuclei, which is based on specific dNTP-binding to 3'-hydroxyl ends of DNA. Here, we report a simple and rapid flow-cytometric method for simultaneous two-color staining of membrane determinants combined with an improved technique for TUNEL in situ. In developing this procedure, we used a well-studied model of apoptosis, the induction of programmed cell death in immature thymocytes by glucocorticoid hormones. Six week.old female BALB/c mice were injected intraperitoneally with 25 mg kg-'n body weight of the synthetic glucocorticoid agonist dexamethasone (FortecortinTM, Merck). Control animals received no treatment. Animals were killed by cervical dislocation 10 h after injection and the thymic tissue removed and placed in ice.cold PBS pH 7.2. The tissue was minced with scissors and then passed through 100-mesh sieves into ice-cold PBS. Cell suspensions were filtered through a 200-mesh sieve and separated by centrifugation at 500g (5 min, 4°C). The cell pellet was resuspended in ice-cold PBS and centrifugation repeated. 106 cells were re.suspended in 100 Izl PBS, 1% bovine serum albumin BSA and incubated (30 min, 4°C) with 100 p.!of a 1:100dilutionof R - p h y c o ~ - ( P E ) conjugated anti-mouse-CD4 antibody (Becton Dickinson). Cells were washed once, resuspended in 100 izl PBS, 1% BSA and incubated (30 min, 4°(3) with 100 p.! of a (a) (b) 1:1000 dilution of biotinylated anti-mouseCD8 antibody (Becton Dickinson). AfterwashI i ing, cells were fixed in 200 p.l 2°6 paraformaldehyde (freshly prepared by dissolving) in PBS, for 30 min at room temperature on a horizontal shaker. They were then washed once, w-. w.. ^p ! permeabilized by incubating (2 min, 4 ° 0 with 100 p.I 0.1% Triton X-100, 0.1% sodium citrate and then washed twice.The TUNELreactionwas I1" . . . . m l " ..... I "'~;'"1 ...... log . . . . . l o 1 " . . . . . I1l l " 1 . . . . I• I I . . . . . II I ii I ii I 04 l, m4 carried out by incubating cells in a moist chaFITC-dUT]P flTC-dtmP mber (1 h, 3700 with 0.3 nmol FITC-12-dU'I~ (d) (c) (Boehringer Mannheim), 3 nmol dATP, 2 Izl • • • • I I . • liB. • lB. • I | . • IIII • • . • i | . • I I . . Ill.. Ill.. l i b 25 m i CoCIz, 25 U ToT (Boeh'lnger Mannhelm) - " 1 !..,,.... ~ ¢ . ; ~ = - - - , ~. ,'" ~ , ,and TdT buffer (30 m i Tris pH7.2, 140 mm ' • "," ",~',~, It" I , sodium cacodylate) in a total reaction volume ,.'~ .~ ,~;,"~.,, ,. ,.'~, . of 50 ~l, The reaction was stopped by adding •....,.:,.,..,, !..'," 2 p,I 0.5 m EDTA. Ceils were washed and the biotlnylated antl-CD8 detected by incubating for 30 rain with 30 p,l of a 1:200 dilution of , s ~ p t a v t d i n - o / c h r o m e (Cy) ( P M r m i ~ n ) , After ii II

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a FACScan (Becton Dickinson) with an argon laser (488 nm) and/or resuspended in Geivatol (Monsanto Chemicals), 2,5% 1,4-dlazobicyclo[2.2.2]-octan (DABCO) (Sigma), transferred to slides and analysed using a Zeiss LSM 10 laserscan microscope. Digitalimages of fluorescence in response to excitation with the helium neon laser (543 nm) for analysis of cells stained with PE and Cy, and the argon Laser (488 nm) for FITC-stained cells were collected at a scan rate of 2 s per image. Digitalimages were stored on a hard disk, transferred to a high-resolution RGB color video Photomonitor (Lucius and Baer) and photographed using Fujicolor Super HG 200 film. Cells were washed in ice-cold PBS, 1°,6BSA; antibodies and conjugate were optimally diluted in PBS, 1% BSA.Assays were performed in 96-well V-bottom microtiter plates. FACS analysis revealed that in dexamethasone-treated animals, 40.8°6of cells were FITC-dUTP positive, i.e. undergoing apoptosis, compared with 1.3%of cells in untreated

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l~ca.I 1. FACSanalysis of thymocytesfrom a BALB/cmouse. The three panels to the left show results for ceils from a dexamethasone-treated mouse, the three to the right, ceils from an untreated animal. Quantitative analysis reveals 40.8% apoptotic cells after dexamethasone treatment (a,b). (c,d) show PE-CD4 staining versus TUNEL (FITC-dtrrP), (e,f) Cy-CD8 staining versus TUNEL(FITC-dtYrP). Note that only 1.3%of thymoo/tes of the untreated animal are apoptotic (Ap).

TIG FEBRUARY1994 VOL. 10 No. 2

41

TECHNICAL

TIPS

controls (Fig. la,b). Dot-plot representation clearly shows that compared with non-apoptotic cells, apoptotic cells are smaller and stain more brightly with FITC-dLv~P(data not shown). Most apoptotic cells were in an immature PE-CD~I+--Cy-CD8+stage of differentiation; a relatively hi/~h percentage of FITC-
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ACKNOWLEDGEMENT This work was supported by the Austrian Research Council (project no. 8809). We thank H. Lassmann for stimulating discussions. REFERENCES 1 Cohen, J.J. (1993) lrnmunol. Today 14, 126-130 2 Darzynkiewicz, Z, el al. (1992) Cytometry 13, 795--808 3 Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992)J. CelIBtol. 119, 493-501 4 Carson, D.A. and Ribeiro, J.M. (1993) Lancet 341, 1251-1254

Contributed by Roswltha Ssonc, Guentber Boeck, Hermann Dietrich,J~b.qnn Gruber, Hetdrun Recbeisand Georg Wick, Institute for Generaland F2@erlmentui Patbolo&v,Univers#yof lnnsim~ck MedicalSchool, Feltz-PreslStrasse3, A-6020 Innsbruck, AusMa.

MONITOR A 'digest' of some recent papers of interest in the primary journals.

OIIgomerlzationof NTRCat the&lnA enhancer Is requiredfor transcriptional activation S.C. PORTER,A.K.NORTII,A.B.WEDEL ANDS. KUSTU Genes Dev. 7, 2258-2273 The expression of one class of lmcterial genes is regulated by a mechanism showing many similarities to that of transcription initiation in eukaryotes. Bacterial genes whose expression requires the 054 factor additk)nally need a spectfic activator protein that binds to enhancer sequences located mote than 100 kb upstream from the promuter. For example, the nitrogen regtllatory protein NTRC activates glnA expression in Salmonella tjpbtmutYmn

by binding to an upstream enhancer. Porter et aL have carried out a careful study of the binding and its effect on transcr/ptional activation of glnA. The enhancer contains two binding sites for NTRC, sepaoted by three turns of the DNA helix. It was known that the

role of these two sites is to concentrate NTRC near the promoter, with which the enhancer interacts by DNA looping, and thereby stimulate formation of an open tonscriptional complex with ILNA polymerase a54. By quantitative binding and gel-shift experiments, Porter et al. show that NTRC binds the glnA enhancer as two directs, that binding is coopeotive, and that phosphorylation of NTRC enhances this coopeotivity at least 12-fold. They also show that the coopeotivity results from inteoction between the two bound dimet~ other than frtan conformatkmal changes in DNA, and that oligomer formation is required for tonscriptional activation, A detailed understanding of inteoctions between enhancer-bound NTRC and the promoter will help to elucidate how NTRC acts as a 'molecular machine', coupling hydrolysis of ATP to a conformational change of 054 holoenzyme and thus inducing formation of an open transcriptional complex, ah TIG FEBRUARY1994 VOL. 10 NO. 2

42

A gene with.specific and global effects on recombination of sequences

from tandemlyrepeatedgenes in

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R.L.KEILANDA.D.MCWILLIAMS Genetics 135, 711-718

Tandemly repeated coding sequences pose special problems for the maintenance of genomic function and integrity: on the one hand, a certain degree of recombination may be necessary to maintain sequence homogeneity (and thus function) in the face of evolutionary drift, but this recombination must be prevented from altering the degree of repetition of the sequence. Keil and McWilliams argue that, if specific genetic functions are involved in achieving this balance, it should be possible to find mutants in those functions, and the),- have identified one mutation (rrm3) that specifically increases recombination of tandemly repeated genes (e.g. rDNA genes) without affecting recombination between