4 | BioProbes 58 | April 2009© 2009 Life Technologies Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these prod-ucts you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

F E AT U R E

At the final stage of apoptosis, adherent cells often detach from the slide or well before they can be detected. Because cells are lost in this way,

you may not be able to detect the DNA fragmentation that is a hallmark, as well as the ultimate determinant, of apoptosis.1 Since its introduction

in 1992,2 the most widely used in situ test for studying DNA fragmentation or strand breaks is the terminal deoxynucleotidyl transferase-dUTP nick

end labeling (TUNEL) assay, which is based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3´-OH

ends of fragmented DNA. For a reliable and reproducible TUNEL imaging assay, it is vital not only that the modified nucleotide is an acceptable

substrate for TdT, but also that the detection method is sensitive and avoids any additional loss of cells from the sample. The Click-iT® TUNEL

Imaging Assays meet both of these criteria. The alkyne-modified dUTP used in the Click-iT® TUNEL assay is rapidly incorporated by TdT, provid-

ing sensitive detection that allows you to fix your samples earlier and preserve late-stage apoptotic cells for imaging before they can detach

(Figure 1). Compared with assays that use other modified nucleotides, the fast and reliable Click-iT® TUNEL Imaging Assays can detect a higher

percentage of apoptotic cells under identical conditions in much less time.

tUNel visionDetect DNA FrAgMeNtAtioN with click-it® tUNel ASSAyS.

Figure 1 (above)—DNA strand breaks typical of late-stage apoptosis visualized using the Click-iT® TUNEL Imaging Assay. HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay (Cat. no. C10247) was performed. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (Cat. no. A11008, green fluorescence). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor® 555 goat anti–mouse IgG (Cat. no. A21422, orange fluorescence). Nuclei were stained with Hoechst 33342 (Cat. no. H1399, blue fluorescence). The coverslips were mounted in ProLong® Gold antifade reagent (Cat. no. P36930) before imaging. The cells on the right not only have a high level of caspase-3 activity and DNA strand breaks, but also show a loss of structural integrity consistent with cells undergoing apoptosis.

www.invitrogen.com | 5© 2009 Life Technologies Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these prod-ucts you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

Detect late-stage apoptosis before cells “pop”The Click-iT® TUNEL Imaging Assays use a dUTP modified with an

alkyne, a small, bio-orthogonal functional group that allows the

nucleotide to be more readily incorporated by TdT than other modi-

fied nucleotides are (Figures 2 and 3). The enzymatically incorporated

nucleotide is detected with a click reaction—a copper-catalyzed triaz-

ole formation between an azide and an alkyne (Figure 4). Click reac-

tions have several characteristics: the reaction between the detection

moieties is efficient; no extreme temperatures or solvents are required;

the reaction product is stable; and the components of the reaction

are bioinert, which means that no side reactions occur—the label and

the detection tags react selectively with one another.3–6 This final point

is the greatest advantage of this powerful detection technique; click

chemistry–labeled molecules can be applied to complex biological

samples and detected with unprecedented sensitivity, thanks

Figure 2—Modified nucleotide structures. The alkyne and bromine modifications are significantly smaller than fluorescein.

Figure 3—Comparison of TdT incorporation of several modified nucleotides. A 48-base single-strand DNA primer plus one type of dUTP nucleotide were incubated with 30 units of TdT enzyme for 3 hr at 37°C. The reaction mixtures were applied to a 20% TBE gel. The gel was stained with ethidium bromide and imaged on a FLA 9000 Fuji scanner. The length of the primer extension was measured by comparing it to the standard 25 bp DNA ladder. To calculate the number of dUTPs added per minute, the length in nucleotides was divided by the time of incubation.

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Figure 4—Detection of apoptosis with the Click-iT® TUNEL assay.

H

NNN

Fix andperm cells

TdT incorporation ofEdUTP into dsDNA

strand breaks(60 min)

Wash(5 min) -N = N+ = N –

Wash(5 min)

Nuclear andoptionalstaining

ImageCu(I)

(Alexa Fluor® azide)

CGATAAECT

CGATAAECT

Wash(5 min)

Fluorescent detection of the incorporated

EdU phosphate with click chemistry(30 min)

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6 | BioProbes 58 | April 2009© 2009 Life Technologies Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these prod-ucts you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

F E AT U R E

Product Quantity Cat. no.Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay for microscopy and HCS, 50–100 assays 1 kit C10245

Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay for microscopy and HCS, 50–100 assays 1 kit C10246

Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay for microscopy and HCS, 50–100 assays 1 kit C10247

Straightforward multiplexed analyses of programmed cell deathTo unequivocally establish programmed cell death in any given cell or

tissue model, two or more biomarkers of apoptosis must be multiplexed.

The Click-iT® TUNEL Imaging Assays are available with a choice of several

bright and photostable Alexa Fluor® dyes, ranging from green to far-red

fluorescence, providing unprecedented flexibility when working with

other apoptosis detection reagents that may only be available in green-

or red-fluorescent options. The Click-iT® TUNEL assays have been tested

in HeLa, A549, and CHO K1 cells with a variety of reagents that induce

apoptosis, including staurosporine, and multiplexed with antibody-based

detection of other apoptosis biomarkers such as cleaved poly(ADP-

ribose) polymerase (PARP), cleaved caspase-3, or phosphohistone 2B

(Figure 6). Each Click-iT® TUNEL kit contains all of the components

necessary to accurately and reliably detect DNA fragmentation in adher-

ent cells grown on coverslips or in 96-well microplates. Learn more at

www.invitrogen.com/click. ■

References 1. Gavrieli, Y. et al. (1992) J Cell Biol 119:493–501. 2. Huerta, S. et al. (2007) J Surg Res 139:143–156. 3. Breinbauer, R. and Köhn, M. (2003) Chembiochem 4:1147–1149. 4. Wang, Q. et al. (2003) J Am Chem Soc 125:3192–3193. 5. Rostovtsev, V.V. et al. (2002) Angew Chem Int Ed Engl 41:2596–2599. 6. Kolb, H.C. et al. (2001) Angew Chem Int Ed Engl 40:2004–2021.

Figure 5—TUNEL assay time course comparison showing the percentage of apoptotic cells detected. HeLa cells were treated with 0.5 µM staurosporine for the lengths of time indicated. Fol-lowing fixation and permeabilization, TUNEL assays using either Click-iT® EdUTP or fluorescein dUTP (DeadEnd™ Colorimetric TUNEL System, Promega) were performed according to the manufacturer’s instructions. The percentage of apoptotic cells was calculated based upon the corresponding negative control. Imaging and analysis was performed using a Thermo Scientific Cellomics® ArrayScan® II (Thermo Fisher Scientific).

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Figure 6 —Click-iT® TUNEL assays used with antibody-based apoptosis detection. HeLa cells were treated with 0.5 µM staurosporine for the lengths of time indicated. The cells were fixed and permeabilized, followed by treatment with the Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay (Cat. no. C10246). Cleaved PARP was detected with a rabbit anti–cleaved PARP antibody and visualized using an Alexa Fluor® 488 goat anti–rabbit IgG antibody (Cat. no. A11034). Cells exhibiting yellow fluorescence are positive for both cleaved PARP (green fluorescence) and DNA strand breaks (red fluorescence). The images were quantitated using the Thermo Scientific Cellomics® ArrayScan® VTI platform (Thermo Fisher Scientific).

2% TUNEL positive2% cleaved PARP positive

2 hr

8% TUNEL positive11% cleaved PARP positive

3 hr

29% TUNEL positive31% cleaved PARP positive

4 hrNo treatment

<1% TUNEL positive1% cleaved PARP positive

to extremely low background. As a result, when compared with assays

using one of the other modified nucleotides, the Click-iT® TUNEL assay

is able to detect a higher percentage of apoptotic cells under identical

conditions (Figure 5) within 2 hours.