Thermo Fisher Scientific Click-iT TUNEL Alexa Fluor Imaging Assay User guide

Brand
Thermo Fisher Scientific
Model
Click-iT TUNEL Alexa Fluor Imaging Assay
Type
User guide
Click-iT™ TUNEL Alexa Fluor™ Imaging Assay
for Microscopy and HCS
Catalog Numbers C10245, C10246, C10247
Pub.No. MAN0002004 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
Since the introduction of the terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay in 1992 (Gavrieli et al., 1992),
the TUNEL assay is the most widely used in situ test for the study of apoptosis (Huerta et al., 2007). The TUNEL assay is based on
the incorporation of modified dUTPs by the enzyme terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA,
a hallmark as well as the ultimate determinate of apoptosis. The modifications are fluorophores or haptens, including biotin or bromine
which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin
or antibodies, if biotin-dUTP or BrdUTP are used, respectively. Often at late stages of apoptosis, adherent cells are known to detach or
“pop” o. For a reliable and reproducible TUNEL imaging assay, the modified nucleotide must not only be an acceptable substrate for
TdT, but the detection method must also be sensitive without bringing about any additional loss of cells from the sample.
The Click-iT™ TUNEL Alexa Fluor™ Imaging Assays utilize a dUTP modified with an alkyne, a small, bio-orthogonal functional group
that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides (Figure1 and Figure2). Detection
is based on a click reaction (Breinbauer and Köhn, 2003; Wang et al., 2003; Rostovtsev et al., 2002; Kolb et al., 2001), a copper (I)
catalyzed reaction between an azide and alkyne (Figure3). Click chemistry fills the void when methods such as direct labeling or the use
of antibodies are not ecient. The small size of the Alexa Fluor™ azide (MW ∼1,000) compared to that of an antibody (MW ∼150,000)
enables eortless penetration of complex samples with only mild fixation and permeabilization required. As a result, when compared to
assays using other modified nucleotides, the Click-iT™ TUNEL Imaging Assay is fast (complete within 2 hours) and is able to detect a
higher percentage of apoptotic cells under identical conditions (Figure4 and Figure5). Furthermore, the Click-iT™ TUNEL Imaging Assay
allows multiplexing with surface and intracellular biomarker detection (Table1).
Figure1Modified nucleotide structures.
The alkyne and bromine modifications are significantly smaller than fluorescein.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
Figure2Comparison of TdT incorporation of several modified nucleotides.
A 48‑bp oligonucleotide was incubated with 30units of TdT and an equimolar mix of the modified nucleotide with three other nucleotides for
4 hours at room temperature. The TdT reaction products were then analyzed by gel electrophoresis, following application to a 20% TBE pre-cast
gel and subsequently stained with SYBR™ Gold nucleic acid gel stain.
Figure3Detection of apoptosis with the Click-iT™ TUNEL Imaging Assay.
Figure4TUNEL assay comparison—percent positives detected.
HeLa cells were treated with 0.5 µM staurosporine for 4 hours. Following fixation and permeabilization, TUNEL imaging assays were performed
according to the manufacturer’s instructions. The percent positives were calculated based upon the corresponding negative control. Imaging and
analysis was performed using a Thermo Scientific™ Cellomics™ ArrayScan™ II.
2 Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide
Figure5TUNEL assay time course comparison—percent positives detected.
HeLa cells were treated with 0.5 µM staurosporine for the time points indicated. Following fixation and permeabilization, the Click-iT™ TUNEL
Imaging Assay using Click-iT™ EdUTP or fluorescein dUTP (Promega™’s DeadEnd™ Fluorometric TUNEL system) was performed according to the
manufacturer’s instructions. The percent positives were calculated based upon the corresponding negative control. Imaging and analysis was
performed using a Thermo Scientific™ Cellomics™ ArrayScan™ II.
Table1Click-iT™ detection reagent compatibility.
Molecule Compatibility[1]
Qdot™ nanocrystals Use Qdot™ nanocrystals after the Click-iT™ detection reaction.
Fluorescent proteins Use organic dye-based reagents, such as TC-FlAsH™ or TC-ReAsH™
reagents for protein expression detection or anti-GFP rabbit or chicken
antibodies after the Click-iT™ detection reaction.
Phalloidin Click chemistry is not compatible with phalloidin staining of F-actin. To
stain the cytoskeleton, antibodies, such as anti-α-tubulin after the Click-
iT™ detection reaction are recommended.
Organic dyes such as Alexa Fluor™ dyes or fluorescein isothiocyanate
(FITC)
Completely compatible with the Click-iT™ detection reaction.
TC-FlAsH™ or TC-ReAsH™ reagents Detect the tetracysteine (TC) tag with TC-FlAsH™ or TC-ReAsH™ reagents
before the Click-iT™ detection reaction.
[1] Some molecules or detection methods involve components that are unstable in the presence of the copper catalyst used for the Click-iT™ detection reaction.
The Click-iT™ TUNEL Alexa Fluor™ Imaging Assay has been tested in HeLa, A549, and CHO K1 cells with a variety of reagents that
induce apoptosis including staurosporine (Figure6). The Click-iT™ TUNEL Alexa Fluor™ Imaging Assay contains all components needed to
accurately and reliably detect apoptosis on adherent cells grown on coverslips or a 96-well microplate, and includes DNase I to generate
strand breaks as a positive control.
Figure6Dose response of staurosporine using the Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay.
HeLa cells were treated with staurosporine for 18hours at final concentrations ranging from 1 nM to 1 µM. Following fixation and permeabilization,
the Click-iT™ TUNEL Imaging Assay was performed. Data points represent averages from 8 wells, the error bars show standard deviations.
Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide 3
Contents and storage
Component Cat. No. C10245 Cat. No. C10246 Cat. No. C10247 Concentration Storage[1,2]
TdT reaction buer
(Component A) 11mL 11mL 11mL 1X solution
≤–20°C
Protect from light
EdUTP nucleotide
mixture (Component B) 100µL 100µL 100µL 50X solution
TdT (terminal
deoxynucleotidyl
transferase)
*recombinant*
(Component C)
6 x 34µL 6 x 34µL 6 x 34µL 15 U/µL in glycerol
Click-iT™ reaction
buer (Component D)
25mL (contains Alexa
Fluor™ 488 azide)
25mL (contains Alexa
Fluor™ 594 azide)
25mL (contains Alexa
Fluor™ 647 azide) 1X solution
Click-iT™ reaction
buer additive
(Component E)
50mg 50mg 50mg Not applicable
Hoechst™ 33342
(Component F) 18µL 18µL 18µL 10mg/mL solution in
water
DNase I
(deoxyribonuclease I,
Component G)
18µL 18µL 18µL Not applicable
DNase I buer
(Component H) 190µL 190µL 190µL 10X solution
Number of assays: Sucient material is supplied for 1 × 96-well plate or 50 coverslips based on the protocols below.
Approximate fluorescence excitation/emission maxima: Alexa Fluor™ 488 azide: 495/519in nm; Alexa Fluor™ 594 azide: 590/615in nm; Alexa
Fluor™ 647 azide: 650/670in nm; Hoechst™ 33342: 350/461 in nm, when bound to DNA.
[1] When stored as directed, this kit is stable for 1year.
[2] These storage conditions are appropriate when storing the entire kit upon receipt. For optimal storage of each component, see labels on individual components.
Before you begin
Required materials not supplied
• 1X Phosphate-buered saline (PBS, Cat. No. 14190-144 or 14190-250)
• 4% Paraformaldehyde in PBS (fixative)
• 0.25% Triton™ X-100 in PBS (permeabilization reagent)
• 3% Bovine serum albumin in PBS (3% BSA in PBS), pH 7.4
• Molecular biology grade water (DNase/RNase free)
• 96-well plate (as recommended for the specific, automated imaging instrument)
• Coverslips for standard microscopy (22mm × 22mm or 18mm × 18mm)
Experimental protocol for cells grown on coverslips
The following protocol was developed using HeLa cells treated with 0.5 µM staurosporine for 4 hours to induce apoptosis. Cell type and
treatment may influence the number of apoptotic cells detected.
This protocol is optimized with a fixation step using 4% paraformaldehyde in PBS followed by a
permeabilization step with 0.25% Triton™X-100, but is amenable to other fixation and permeabilization
reagents such as 70% ethanol.
1.1. Remove media and wash coverslips once with PBS.
Note: If there is a chance that cells may be lost by this wash step, proceed directly to fixation
(step1.2) without performing the wash step.
1.2. Add a sucient volume of fixative (4% paraformaldehyde) to completely cover the coverslips.
1Fix and permeabilize
cells
4 Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide
1Fix and
permeabilize cells
(continued)
1.3. Incubate samples for 15 minutes at room temperature.
1.4. Remove fixative.
1.5. Add sucient volume of the permeabilization reagent (0.25% Triton™ X-100 in PBS) to
completely cover the coverslips.
1.6. Incubate samples for 20 minutes at room temperature. Then wash twice with deionized water.
The DNase I generates strand breaks in the DNA to provide a positive TUNEL reaction. There is
sucient DNase I to perform ∼18 positive controls.
2.1. Wash coverslips with deionized or molecular biology grade water.
2.2. Prepare DNase I solution according to Table2 and mix well.
IMPORTANT! Do not vortex the DNase I solution as DNase I denatures with vigorous mixing.
Table2DNase I solution.
Reaction components
Number of coverslips
1 2 3
Deionized water 89µL 178µL 267µL
DNase I buer
(ComponentH) 10µL 20µL 30µL
DNase I (ComponentG) 1µL 2µL 3µL
Total volume 100µL 200µL 300µL
2.3. Add 100 µL of the DNase I solution (prepared in step2.2) to each coverslip and incubate for
30 minutes at room temperature.
2.4. Wash coverslips once with deionized water, proceed to “Perform the TdT reaction” on page5.
2(Optional) Prepare a
positive control
This protocol uses 100 µL of the TdT reaction cocktail per coverslip. It is important to equilibrate cells
to maximize eciency of the TdT reaction.
3.1. Add 100 µL TdT reaction buer (Component A) to each coverslip and allow the solution to
spread completely over the surface.
3.2. Incubate coverslips for 10 minutes at room temperature.
3.3. Remove TdT reaction buer.
3.4. Prepare the TdT reaction cocktail according to Table3.
Table3TdT reaction cocktails.
Reaction
components
Number of coverslips
1 2 4 5 10
TdT reaction
buer
(ComponentA)
94µL 188µL 376µL 470µL 940µL
EdUTP
(ComponentB) 2µL 4µL 8µL 10µL 20µL
TdT[1]
(ComponentC) 4µL 8µL 16µL 20µL 40µL
Total volume 100µL 200µL 400µL 500µL 1mL
[1] Immediately after using the TdT enzyme (Component C), return the vial ≤–20°C. Do not keep the enzyme at room
temperature.
3Perform the TdT
reaction
Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide 5
3Perform the TdT
reaction (continued)
3.5. Add 100 µL of the TdT reaction cocktail (prepared in step3.4) to each coverslip and allow the
solution to spread completely over the surface.
3.6. Incubate coverslips for 60 minutes at 37°C. Use a humidified chamber to protect against
evaporation. Remove the reaction cocktail.
Note: This reaction can be performed overnight at room temperature.
3.7. Wash coverslips twice with 3% BSA in PBS for 2 minutes each.
4.1. Prepare the Click-iT™ reaction buer additive (Component E) by adding 625 µL of deionized
water to the Component E vial. After use, aliquot and store any remaining solution at ≤–20°C.
When stored as directed, the stock solution is stable for up to 1 year.
4.2. Prepare the Click-iT™ reaction cocktail according to Table4 and mix well by vortexing.
IMPORTANT! Use the Click-iT™ reaction cocktail within 15 minutes of preparation.
Table4Click-iT™ reaction cocktails.
Reaction
components
Number of coverslips
1 2 4 5 10
Click-iT™
reaction buer
(ComponentD)
97.5µL 195µL 390µL 487.5µL 975µL
Click-iT™
reaction buer
additive
(prepared in
step4.1)
2.5µL 5µL 10µL 12.5µL 25µL
Total volume 100µL 200µL 400µL 500µL 1mL
4.3. Immediately add 100µL of the Click-iT™ reaction cocktail (prepared in step4.2) to each coverslip
and allow the solution to spread completely over the surface.
4.4. Incubate coverslips for 30 minutes at room temperature, protected from light.
4.5. Remove the Click-iT™ reaction cocktail and wash each coverslip with 3% BSA in 1X PBS for
5 minutes.
For antibody staining, proceed to “(Optional) Stain with antibodies” on page6, or for DNA
staining, proceed to “(Optional) Stain DNA” on page7. If no additional staining is desired,
proceed to “Image and analyze” on page7.
4Perform the Click-iT™
reaction
5.1. If required, block the coverslips or wells with 3% BSA in 1X PBS for the recommended time,
protected from light. Remove the blocking solution.
5.2. Prepare and add the primary antibody solution as recommended by the manufacturer.
5.3. Incubate for the recommended time and temperature, protected from light. Remove the
primary antibody solution.
5.4. Wash each coverslip or well twice with 3% BSA in PBS. Remove the wash solution.
5.5. Prepare and add the secondary antibody solution as recommended by the manufacturer.
5.6. Incubate for the recommended time and temperature, protected from light. Remove the
secondary antibody solution.
5.7. Wash each coverslip or well twice with 3% BSA in PBS. Remove the wash solution.
For DNA staining, proceed to “(Optional) Stain DNA” on page7. If no additional staining is
desired, proceed to “Image and analyze” on page7.
5(Optional) Stain with
antibodies
6 Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide
6.1. Dilute Hoechst™ 33342 (Component F) 1:5,000 in PBS to obtain a 1X Hoechst™ 33342 solution.
6.2. Add 100µL 1X Hoechst™ 33342 solution per coverslip or well and incubate for 15 minutes at
room temperature, protected from light. Remove the Hoechst™ 33342 solution.
6.3. Wash each coverslip or well twice with PBS. Remove the wash solution.
6(Optional) Stain DNA
The Click-iT™ TUNEL stained cells are compatible with all methods of slide preparation including wet
mount or prepared mounting media. See Table5 for the approximate fluorescence excitation/emission
maxima for Alexa Fluor™ dyes and Hoechst™ 33342 bound to DNA.
Table5Approximate fluorescence excitation/emission maxima.
Fluorophore Excitation (nm) Emission (nm)
Alexa Fluor™ 488 495 519
Alexa Fluor™ 594 590 615
Alexa Fluor™ 647 650 670
Hoechst™ 33342 bound to DNA 350 460
7Image and analyze
Experimental protocol for cells grown in a 96-well microplate
The following protocol was developed using HeLa cells treated with 0.5 µM staurosporine for 4 hours to induce apoptosis. Cell type and
treatment may influence the number of apoptotic cells detected.
This protocol is optimized with a fixation step using 4% paraformaldehyde in PBS followed
by a permeabilization step using 0.25% Triton™ X-100, but is amenable to other fixation and
permeabilization reagents such as 70% ethanol.
1.1. Remove media and wash wells once with PBS.
Note: If there is a chance that cells maybe lost by this wash step, proceed directly to fixation
(step1.2) without performing the wash step.
1.2. Add 100µL fixative (4% paraformaldehyde) to completely cover each well.
1.3. Incubate plates for 15 minutes at room temperature.
1.4. Remove fixative.
1.5. Add 100 µL of the permeabilization reagent (0.25% Triton™ X-100 in PBS) to completely cover
each well.
1.6. Incubate plates for 20 minutes at room temperature.
1.7. Wash each well twice with deionized water.
1Fix and permeabilize
cells
The DNase I creates strand breaks in the DNA to provide a positive TUNEL reaction. There is sucient
DNase I to perform ∼18 positive control wells.
2(Optional) Prepare a
positive control
Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide 7
2(Optional) Prepare
a positive control
(continued)
2.1. Wash the wells with deionized or molecular biology grade water.
2.2. Prepare DNase I solution according to Table6 and mix well.
IMPORTANT! Do not vortex the DNase I solution as DNase I denatures with vigorous mixing.
Table6DNase I solution.
Reaction
components
Number of wells
1 2 4 5 8
Deionized water 89µL 178µL 356µL 445µL 712µL
DNase I buer
(ComponentH) 10µL 20µL 40µL 50µL 80µL
DNase I
(ComponentG) 1µL 2µL 4µL 5µL 8µL
Total volume 100µL 200µL 400µL 500µL 800µL
2.3. Add 100 µL to each well that will be a positive control and incubate for 30 minutes at room
temperature.
2.4. Wash each well once with deionized water and proceed to “Perform the TdT reaction” on
page8.
This protocol uses 50 µL of the TdT reaction cocktail per well. It is important to equilibrate the cells to
maximize the eciency of TdT reaction.
3.1. Add 50 µL of the TdT reaction buer (Component A) to each well and allow the solution to
spread completely over the surface.
3.2. Incubate plates for 10 minutes at room temperature. Remove the TdT reaction buer.
3.3. Prepare the TdT reaction cocktail according to Table7.
Table7TdT reaction cocktails.
Reaction
components
Number of wells
1 8 12 100
TdT reaction buer
(ComponentA) 47µL 376µL 564µL 4.7mL
EdUTP
(ComponentB) 1µL 8µL 12µL 100µL
TdT[1]
(ComponentC) 2µL 16µL 24µL 200µL
Total volume 50µL 400µL 600µL 5mL
[1] Immediately after using the TdT enzyme (Component C), return the vial ≤–20°C. Do not keep the enzyme at room
temperature.
3.4. Add 50 µL of the TdT reaction cocktail (prepared in step3.3) to each well and allow the solution
to spread evenly in the well.
3.5. Incubate plates for 60 minutes at 37°C. Remove the reaction cocktail.
3.6. Wash wells twice with 3% BSA in PBS for 2 minutes each.
3Perform the TdT
reaction
8 Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide
4.1. Prepare the Click-iT™ reaction buer additive (Component E) by adding 625 µL of deionized
water. After use, aliquot and store any remaining solution at ≤–20°C. When stored as directed,
the stock solution is stable for up to 1 year.
4.2. Prepare the Click-iT™ reaction cocktail according to Table8 and mix well by vortexing.
IMPORTANT! Use the Click-iT™ reaction cocktail within 15 minutes of preparation.
Table8Click-iT™ reaction cocktails.
Reaction
components
Number of wells
1 8 12 100
Click-iT™
reaction buer
(ComponentD)
97.5µL 780µL 1.17mL 9.75mL
Click-iT™ reaction
buer additive
(prepared in
step4.1)
2.5µL 20µL 30µL 250µL
Total volume 100µL 800µL 1.2mL 10mL
4.3. Immediately add 50µL of the Click-iT™ reaction cocktail (prepared in step4.2) to each well and
allow the solution to spread completely over the surface.
4.4. Incubate plates for 30 minutes at room temperature, protected from light. Remove the reaction
cocktail.
4.5. Wash each well with 3% BSA in 1X PBS for 5 minutes.
4.6. (Optional) Stain with antibodies or other stains such as DNA staining with Hoechst™ 33342 (see
“(Optional) Stain with antibodies” on page6 and “(Optional) Stain DNA” on page7).
4.7. Image plate using filters appropriate for the Alexa Fluor™ dye included in the Click-iT™ TUNEL
Imaging Assay (Table5) and any other fluorophores used.
4Perform the Click-iT™
reaction
Related products
Product Cat. No. Unit size
Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay, for Microscopy and HCS C10245 50–100 assays
Click-iT™ TUNEL Alexa Fluor™ 594 Imaging Assay, for Microscopy and HCS C10246 50–100 assays
Click-iT™ TUNEL Alexa Fluor™ 647 Imaging Assay, for Microscopy and HCS C10247 50–100 assays
Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 488 dye C10617 50 assays
Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 594 dye C10618 50 assays
Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 647 dye C10619 50 assays
Click-iT™ TUNEL Colorimetric IHC Detection Kit C10625 50 assays
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please
contact Life Technologies at www.thermofisher.com/support.
References
Gavrieli Y, Sherman Y, Ben-Sasson SA. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J Cell Biol. 119:493–501.
Click-iT™ TUNEL Alexa Fluor™ Imaging Assay User Guide 9
Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH. (2007) Screening and detection of apoptosis. J Surg Res. 139:143–156.
Breinbauer R, Köhn M. (2003) Azide-alkyne coupling: a powerful reaction for bioconjugate chemistry. Chembiochem. 4:1147–1149.
Wang Q, Chan TR, Hilgraf R, Fokin VV, Sharpless KB, Finn MG. (2003) Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2]
cycloaddition. J Am Chem Soc. 125:3192–3193.
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. (2002) A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective
"ligation" of azides and terminal alkynes. Angew Chem Int Ed Engl. 41:2596–2599.
Kolb HC, Finn MG, Sharpless KB. (2001) Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed
Engl. 40:2004–2021.
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0002004
Revision Date Description
A.0 15 April 2022 The format and content were updated. The version numbering was reset to A.0 in
conformance with internal document control.
1.00 14 November 2008 New document for the Click-iT™ TUNEL Alexa Fluor™ Imaging Assay.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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Thermo Fisher Scientific Click-iT TUNEL Alexa Fluor Imaging Assay User guide

Brand
Thermo Fisher Scientific
Model
Click-iT TUNEL Alexa Fluor Imaging Assay
Type
User guide
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