Tunel说明书

in diagnostic procedures. FOR IN VITRO USE ONLY.

In Situ Cell DeathDetection Kit, TMR red

Version April 2006

Kit for detection and quantification of apoptosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by fluorescence microscopy or flow cytometry.

Cat. No. 12 156 792 9101 Kit (50 tests)

Store at Ϫ15 to Ϫ25°C

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1. Preface

1.1 Table of Contents

1. Preface ............................................................................................................ 21.1 Table of Contents 21.2 Kit Contents 32. Introduction ................................................................................................... 52.1Product Overview 52.2 Background Information 83. 3.13.23.2.13.2.2 3.2.33.2.3.1 3.2.3.2 3.33.3.1 3.3.2 3.3.33.3.44.

Procedures and Required Materials ...................................................... 10Flow Chart 10Preparation of Sample Material 11Cell Suspension 11Adherent Cells, Cell Smears, and Cytospin Preparations 12Tissue Sections 13Treatment of Paraffin-Embedded Tissue 13Treatment of Cryopreserved Tissue 15Labeling Protocol 16Before you Begin 16Labeling Protocol for Cell Suspensions 17Labeling Protocol for Adherent Cells, Cell Smears, Cytospin Preparations,and Tissues 18Labeling Protocol for Difficult Tissue 19Typical results ............................................................................................. 20

5.Appendix ....................................................................................................... 21

5.1Troubleshooting 215.2 References 245.3 Ordering Information 25

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1.2 Kit ContentsCaution

The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.

Avoid exposure and obtain special instructions before use.

When using do not eat, drink or smoke. After contact with skin, wash immediately with plenty of water. In case of accident or if you feel unwell seek medical advice immediately (show label where possible).Collect the supernatants from the labeling reactions in a tightly closed, non-breakable container and indicate contents. Discard as regulated for toxic waste.

Please refer to the following table for the contents of the kit.Vial/Cap1blue

Label

Contents

Kit Contents

Enzyme Solution•Terminal deoxynucleotidyl transferase

from calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5 × 50 ␮lLabel Solution

•Nucleotide mixture in reaction buffer•1× conc.•5 × 550 ␮l

2red

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1.2 Kit Contents, continued

Additional Solutions Required

Procedure

In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.

Detailed information is given in front of each procedure.

Equipment

Reagents

Preparation of sample material (section 3.2)

•Cell suspension •Shaker•Washing buffer: Phosphate buffered (section3.2.1)•V-bot-saline (PBS*)•Adherent cells, cell smears tomed 96-•Fixation solution: 4% Paraformaldehyde in and cytospin preparations well micro-PBS, pH 7.4, freshly prepared(section 3.2.2.)plate•Permeabilisation solution: 0.1% Triton X-•Cryopreserved tissue 100 in 0.1% sodium citrate, freshly pre-(section 3.2.3.2)pared (6)Paraffin-embedded tissue (section 3.2.3.1)

•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)

•Washing buffer: PBS*

•Proteinase K*, nuclease free, working solution: [10–20 ␮g/ml in 10 mM Tris/HCl, pH 7.4–8]

Alternative treatments•Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate), freshly pre-pared

•Pepsin* (0.25%–0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free

•0.1 M Citrate buffer, pH 6 for microwave irradiation•Micrococcal nuclease or •DNase Irecombinant*

•Parafilm or Washing buffer: PBS*

coverslips•Humidified chamber

•Plastic jar•Citrate buffer, 0.1 M, pH 6.0.•Microwave•Washing buffer: PBS*

•Humidified •Tris-HCl, 0.1 M pH 7.5, containing 3% chamberBSA* and 20% normal bovine serum

Labeling protocol (section 3.3)Positive control

(section 3.3.1)•Cell suspensions (section3.3.2)•Adherent cells (section3.3.3)Difficult tissue(section 3.3.4)

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2. Introduction

2.1

Product Overview

Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks“) in high molecular weight DNA.

Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Stage1DescriptionLabeling of DNA strand breaks by Terminal deoxynucleotidyl transferase (TdT) which catalyzes polymerization of labeled nucleotides to free 3’-OH DNA ends in a template-indepen-dent manner (TUNEL-reaction).TMR red labeled nucleotides, incorporated in nucleotide poly-mers, are detected and quantified by fluorescence microscopy or flow cytometry.Test Principle

2Fig. 1: DNA of fixed cells labeled by the addition of TMR red dUTP at strandbreaks by terminal transferase.

Application

The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic cell death at single cell level in cells and tissues. Thus, the In Situ Cell Death Detection Kit can be used in many different assay systems. Examples are:

•Detection of individual apoptotic cells in frozen and formalin fixed tissue sections in basic research.

•Determination of sensitivity of malignant cells to drug induced apop-tosis in cancer research.

•Typing of cells undergoing cell death in heterogeneous populations by double staining procedures (6, 7).

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2.1Product Overview, continued

Specificity

The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis from necrosis and from primary DNA strand breaks induced by cytostatic drugs or irradiation (3, 4).

Test InterferenceFalse negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such as extracellular matrix components can prevent access of TdT to DNA strand breaks. In either case false negative results may be obtained.False positive results: Extensive DNA fragmentation may occur in cer-tain forms of necrosis (38).

DNA strand breaks may also be prominent in cell populations with high proliferative or metabolic activity. In either case false positive results may be obtained.

To confirm apoptotic mode of cell death, the morphology of respective cells should be examined very carefully. Morphological changes dur-ing apoptosis have a characteristic pattern. Therefore evaluation of cell morphology is an important parameter in situations where there is any ambiguity regarding interpretation of results.Sample Material

•Cell suspensions from

•permanent cell lines (2, 27, 35),

•lymphocytes and leukemic cells from peripheral blood (4),•thymocytes (1, 6),•bone marrow cells

•fine needle biopsies (5)

•Cytospins and cell smear preparations

•Adherent cells cultured on chamber slides (31)

•Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25, 26, 29, 30, 32–34, 36, 39)1-2 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.The kit is designed for 50 tests.

The unopened kit is stable at Ϫ15 to Ϫ25°C through the expiration date printed on the label.

Note: The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.

Please refer to the following table.

BenefitSensitive

Feature

Detection of apoptotic cell death at single cell level via fluorescence microscope and at cell pop-ulations via FACS analysis at very early stages (1, 2, 6).

Assay TimeNumber of TestsKit Storage/ Stability

Advantage

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2.1Product Overview, continued

SpecificFastConvenient

Preferential labeling of apoptosis versus necrosis (3, 4).

Short assay time (1–2 h).

•No secondary detection system required.•One incubation and one washing step only.•Reagents are provided in stable, optimized form.

•No dilution steps required.

•Application in combination with fluorescein label possible•Suitable for fixed cells and tissue. This allows accumulation, storage and transport of sam-ples (2, 5).

•Double staining enables identification of type and differentiation state of cells undergoing apoptosis (6).Every lot is function-tested on apoptotic cells in comparison to a master lot.

Flexible

Function-tested

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2.2 Background InformationCell Death

Two distinct modes of cell death, apoptosis and necrosis, can be dis-tinguished based on differences in morphological, biochemical and

molecular changes of dying cells.

Programmed cell death or apoptosis is the most common form of eukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normal tissue turnover (8, 9). In general, cells undergoing apoptosis display a characteristic pattern of structural changes in nucleus and cytoplasm, including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage to chromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been described where morphological features of apoptosis are not accompanied with oligonucleosomal DNA cleavage (37).

Apoptosis is essential in many physiological processes, including maturation and effector mechanisms of the immune system (12, 13), embryonic development of tissue, organs and limbs (14), development of the nervous system (15, 16) and hormone-dependent tissue remodeling (17). Inappropriate regulation of apoptosis may play an important role in many pathological conditions like ischemia, stroke, heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).

In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumor drugs, radiation and hyperthermia, and that the intrinsic propensity of tumor cells to respond by apoptosis is modulated by expression of several oncogenes and may be a prognostic marker for cancer treat-ment (21).

Apoptosis

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continued2.2 Background Information,

Identification of

Apoptosis

Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder” on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation. This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks.DNA polymerase as well as terminal deoxynucleotidyl transferase (TdT) (1–6, 25–36) have been used for the incorporation of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:

•Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4).

•Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).

•TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).

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Required Materials3. Procedures and

The working procedure described below was published by R. Sgonc

and colleagues (6). The main advantage of this kit is the use of tetra-methyl- rhodamine- dUTP to directly label DNA strand breaks with red fluorescence. This allows the direct detection of DNA fragmentation in the red channel and secondary labeling with fluorescein in the green channel by flow cytometry or fluorescence microscopy.

3.1

Flow Chart

The assay procedure is explained in the following flow chart.

Adherent cells, cell smears, and cytospin prepara-tions

↓Fixation↓

↓

Labeling reaction

with TUNEL reaction mixture

↓Analyze by flow cyto-metry or by fluores-cence microscopy

↓↓

Permeabilisation

Cryopre-served tissue sections

↓

Paraffin-embedded tissue sections

↓

Dewaxation RehydrationProtease treat-ment

↓

Assay Procedure

Cell suspension

↓

Analyze by fluorescence microscopy

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3.23.2.1

Preparation of Sample MaterialCell Suspension

For dual parameter flow cytometry with fluorescein-conjugated anti-bodies, incubate the cells prior to fixation with the cell surface marker.•Washing buffer: Phosphate buffered saline (PBS)

•Fixation solution: Paraformaldehyde (4% in PBS, pH 7.4), freshly pre-pared

•Permeabilisation solution: 0.1% Triton X–100 in 0.1% sodium citrate, freshly prepared•Shaker

•V-bottomed 96-well microplate

Note: Use of a V-bottomed 96-well microplate minimize cell loss dur-ing fixation, permeabilisation and labeling and allows simultaneous preparation of multiple samples.

Please find in the following protocol the procedure for cell fixation and permeabilisation.

Note: Fix and permeabilisate two additional cells for the negative and positive labeling controls.Step1234

Action

Wash test sample 3 times in PBS and adjust to 2 × 107 cells/ml.Transfer 100 ␮l/well cell suspension into a V-bottomed 96-well microplate.

Add 100 ␮l/well of a freshly prepared Fixation solution to cell suspension (final concentration 2% PFA).

Resuspend well and incubate 60 min at +15 to +25°C.

Note: To avoid extensive clumping of cells, microplate should be incubated on a shaker during fixation.

Centrifuge microplate at 300 g for 10 min and remove fixative by flicking off or suction.

Wash cells once with 200 ␮l/well PBS.

Centrifuge microplate at 300 g for 10 min and remove PBS by flicking off or suction.

Resuspend cells in 100 ␮l/well Permeabilisation solution for 2 min on ice (+2 to +8°C).Proceed as described under 3.3.

PrelabelingAdditional Buff-ers and Equip-ment Required

Procedure

567

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3.2.2 Adherent Cells, Cell Smears, and Cytospin Preparations

•Washing buffer: Phosphate buffered saline (PBS)

•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared

•Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly prepared (6)The following table describes preparations of adherent cells, cell smears and cytospin.

Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step1234

Action

Fix air dried cell samples with a freshly prepared Fixation solution for 1 h at +15 to +25°CRinse slides with PBS.

Incubate in Permeabilisation solution for 2 min on ice (+2 to +8°C).

Proceed as described under 3.3.

Additional Solutions Required

Procedure

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3.2.3Tissue Sections

3.2.3.1 Treatment of Paraffin-Embedded TissuePretreatment of Paraffin Embed-ded Tissue

Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 41).

Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!

The other 3 alternative procedures are also described in the following table (step 2).

•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water)•Washing buffer: PBS

•Proteinase K, PCR grade*, working solution: [10-20 ␮g/ml in 10 mM Tris/HCl, pH 7.4-8]Alternative treatments•Permeabilisation solution: 0.1% Triton X–100, 0.1% sodium citrate, freshly prepared

•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free

•0.1 M Citrate buffer, pH 6 for the microwave irradiation

Additional Solutions Required

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3.2.3.1 Treatment of Paraffin-Embedded Tissue, continued

Procedure

In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures are described.

Note: Add additional tissue sections for the negative and positive labeling controls.Step1

Action

Dewax and rehydrate tissue section according to standard protocols (e.g., by heating at +60°C followed by washing in xylene and rehydration through a graded series of ethanol and double dist. water) (1, 33, 36).

Incubate tissue section for 15–30 min at +21 to +37°C with Proteinase K working solution.

Alternatives:1. Permeabilisa-tion solution

Treatment:

Incubate slides for 8 min.

2

2. Pepsin* (30, 40) 15–60 min at +37°C.or trypsin*

3. Microwave irra-•Place the slide(s) in a plastic jar diationcontaining 200 ml 0.1 M Citrate

buffer, pH 6.0.

•Apply 350 W microwave irradia-tion for 5 min.

34

Rinse slide(s) twice with PBS.Proceed as described under 3.3.

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3.2.3.2 Treatment of Cryopreserved TissueAdditional Solutions required

•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared

•Washing buffer: PBS

•Permeabilisation solution: 0.1% Triton X–100, 0.1% sodium citrate, freshly preparedIn the following table the pretreatment of Cryopreserved tissue is described.

Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step12

Action

Fix tissue section with Fixation solution for 20 min at +15 to +25°C.

Wash 30 min with PBS.

Note: For storage, dehydrate fixed tissue sections 2 min in absolute ethanol and store at Ϫ15 to Ϫ25°C.

Incubate slides in Permeabilisation solution for 2 min on ice (+2 to +8°C).

Proceed as described under 3.3.

Cryopreserved Tissue

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3.33.3.1

Labeling ProtocolBefore you Begin

One pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) is sufficient for staining 10 samples by using 50 ␮l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 ␮l Label Solution per control.

Note: The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.Step12

Action

Remove 100 ␮l Label Solution (vial 2) for two negative con-trols.

Add total volume (50 ␮l) of Enzyme Solution (vial 1) to the remaining 450 ␮l Label Solution in vial 2 to obtain 500 ␮l TUNEL reaction mixture.

Mix well to equilibrate components.

Preparation of TUNEL Reaction Mixture

3

Additional Reagents RequiredControls

•Micrococcal nuclease or •DNase Irecombinant *

Two negative controls and a positive control should be included in each experimental set up.Negative control:Positive control:

Incubate fixed and permeabilized cells in 50 ␮l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.

Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I recombinant (3000U/ml– 3 U/ml in 50 mM Tris-HCl, pH 7.5, 1 mg/ml BSA) for 10 min at +15 to +25°C to induce DNA strand breaks, prior to labeling procedures.

* available from Roche Applied Science

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3.3.2 Labeling Protocol for Cell Suspensions

•Washing buffer: PBS•Humidified chamber

Additional

Equipment and Reagents RequiredProcedure

Please refer to the following table. Step123456

Action

Wash cells twice with PBS (200 ␮l/well).

Resuspend in 50 ␮l/well TUNEL reaction mixture.Note: For the negative control add 50 ␮l Label solution.Add lid and incubate for 60 min at +37°C in a humidified atmosphere in the dark.Wash samples twice in PBS.

Transfer cells in a tube to a final volume of 250-500 ␮l in PBS.Samples can directly be analyzed by flow cytometry or fluo-rescence microscopy. For evaluation by fluorescence micros-copy use an excitation wavelength in the range of 520-560 nm (maximum 540 nm; green) and detection in the range of 570-620 nm (maximum 580 nm, red).

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3.3.3

Labeling Protocol for Adherent Cells, Cell Smears, Cytospin Preparations,and Tissues

Additional •Washing buffer: PBSEquipment and •Parafilm or coverslipsReagents Required

•Humidified chamberProcedure

Please refer to the following table.Step123

Dry area around sample.

Add 50 ␮l TUNEL reaction mixture on sample.

Note: For the negative control add 50 ␮l Label solution each. To ensure a homogeneous spread of TUNEL reaction mixture across cell monolayer and to avoid evaporative loss, samples should be covered with parafilm or coverslip during incuba-tion.

Incubate slide in a humidified atmosphere for 60 min at +37°C in the dark.

Rinse slide 3× with PBS.

Samples can directly be analysed under a fluorescence micro-scope or embedded with antifade prior to analysis. Use an excitation wavelength in the range of 520-560 nm (maximum 540 nm; green) and detection in the range of 570-620 nm (maximum 580 nm, red).

Action

Rinse slides twice with PBS.

456

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3.3.4Labeling Protocol for Difficult Tissue

•Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS

•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum•Plastic jar•Microwave

Please refer to the following table.Step123

Action

Dewax paraformaldehyde- or formalin-fixed tissue sections according to standard procedures.

Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH 6.0.

•Apply 750 W (high) microwave irradiation for 1 min.•Cool rapidly by immediately adding 80 ml double dist. water (+20 to +25°C).

•Transfer the slide(s) into PBS (+20 to +25°C).DO NOT perform a Proteinase K treatment!

Immerse the slide(s) for 30 min at +15 to +25°C in Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum.

•Rinse the slide(s) twice with PBS at +15 to +25°C.•Let excess fluid drain off.

Add 50 ␮l of TUNEL reaction mixture on the section.Note: For the negative control add 50 ␮l Label solution.Incubate for 60 min at +37°C in a humidified atmosphere in the dark.

•Rinse slide(s) three times in PBS for 5 min each.

•Evaluate the section under a fluorescence microscope.

Additional

Equipment and Solutions Required

Procedure

4

5678

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4. Typical results

Assay Procedures•Incubate U937 cells at a density of 106 cells/ml in the presence of

camptothecin (2 ␮g/ml, 4 h at +37°C) to induce apoptosis.

•As control for non-apoptotic population, an aliquot of the cells is incubated in normal culture medium without camptothecin.•Harvest cells and proceed as described under 3.2.1.

Fig. 2: Analysis of camptothecin induced apoptosis in U937 cell by flow cytometryDotted line: Cells cultured in the absence of camptothecin.

Solid line: Cells cultured in the presence of camptothecin (2 ␮g/ml, 4 h).Cells analyzed under marker M1 are apoptotic (TUNEL positive)

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5.

5.1

Appendix

Troubleshooting

This table describes various troubleshooting parameters.

Problem

Step/Reagent of Procedure

Possible Cause

Recommendation

Nonspecificlabeling

Embedding of UV-irradiation forTry different embedding material or tissuepolymerization of different polymerization reagent.

embedding material (e.g., methacrylate) leads to DNA strand breaksFixation

Acidic fixatives (e.g., methacarn, Carnoy’s fixative)TdT concentration too high

•Try 4% buffered paraformaldehyde.•Try formalin or glutaraldehyde.Reduce concentration of TdT by dilu-ting it 1:2 up to 1:10 with TUNEL Dilu-tion Buffer*.

TUNEL reactionNucleases, Polymerases

Some tissues (e.g., •Fix tissue immediately after organ smooth muscles) preparation.

show DNA strand •Perfuse fixative through liver vein.breaks very soon after tissue prepa-ration.

Some enzymes are Block with a solution containing ddUTP still active.and dATP.

High back-ground

Sample

Mycoplasma con-tamination

Mycoplasma Detection Kit*

Highly proliferating Double staining, e.g., with Annexin-V-cellsFluos*.

Note: Measuring via microplate reader not possible because of too high back-ground.

Fixation

Formalin fixation Try methanol for fixation but take into leads to a yellowish account that this might lead to reduced staining of cells sensitivity.containing melanin precursors.Concentration of labeling mix is too high for mamma carcinoma.

Reduce concentration of labeling mix to 50% by diluting with TUNEL Dilution Buffer.

continued on next page

TUNEL reac-tion

* available from Roche Applied Science

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5.1Troubleshooting, continued

Problem

Step/Reagent of Procedure

Possible CauseRecommendation

Low labelingFixation

Ethanol and metha-•Try 4% buffered paraformaldehyde.nol can lead to low •Try formalin or glutaraldehyde.labeling (nucleo-somes are not cross-linked with proteins during fix-ation and are lost during the proce-dure steps)Extensive fixation leads to excessive cross-linking of proteins

•Reduce fixation time.

•Try 2% buffered paraformaldehyde.

Permeabilisa-tion

Permeabilisation too short so that reagents can’t reach their target molecules

•Increase incubation time.

•Incubate at higher temperature (e.g., +15 to +25°C).

•Try Proteinase K (concentration and time has to be optimized for each type of tissue).

•Try 0.1 M sodium citrate at +70°C for 30 min.•Treat tissue sections after dewaxing with Proteinase K (concentration, time and temperature have to be optimized for each type of tissue).•Try microwave irradiation at 370 W (low) for 5 min in 200 ml 0.1 M Cit-rate buffer pH 6.0 (has to be opti-mized for each type of tissue).

continued on next page

Paraffin-embeddingAccessibility for reagents is too low

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5.1Troubleshooting, continued

Problem

Step/Reagent of Procedure

Possible CauseRecommendation

No signal on DNase positive con-treatmenttrol

Concentration of DNase is too low

•For cryosections apply 3 U/ml DNase I recombinant.

•For paraffin-embedded tissue sec-tions apply 1500 U/ml DNase I recombinant.

•In general, use 1 U/ml DNase I recombinant, dissolved in 10 mM Tris-HCl, pH 7.4 containing 10 mM NaCl, 5 mM MnCl2, 0.1 mM CaCl2, 25 mM KCl and incubate 30 min at +37°C.

•Alternative buffer: Tris- HCl pH 7.5 containing 1 mM MgCl2 and 1 mg/ml BSA.

Counter-staining diminishes TUNEL stainingEquivocal signals

DNA stain

Too high concentra-Use 0.1–1 ␮g/ml BoBo–1 from Molec-tions of DNA dyeular Probes for counterstaining.

Double

staining

Earlier stage of apoptosis than stage detected by TUNEL reaction

For additional measurement of apopto-sis: M30 CytoDEATH* is suitable or Annexin V – Fluos*.

Problems with inter-pretation of results

FACS Analysis

Positive and nega-Change apoptosis inducing procedure:tive peaks are not 2-3 Clusters should be visible in the distinguishable, FSC/SSC histogram:because too many 1.debris and apoptotic bodiesapoptotic bodies 2.whole cellsacquired, apoptosis 3.shrinked cells gate should delete is too far1.): clearly separated peaks.No signal for apop-Time depends on cell line and inducing tosisagents and should be optimized.

* available from Roche Applied Science

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5.2 References

12345671011121314151617181920212223242526272829303132333435363738394041Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493-501.

Gorczyca, W., Gong, J. & Darzynkiewicz, Z. (1993) Cancer Res. 53, 1945-1951.

Gorczyca, W. et al. (1993) Leukemia 7, 659-670.Gold, R. et al. (1994) Lab. Invest. 71, 219

Gorczyca, W. et al. (1994) Cytometry 15, 169-175.Sgonc, R. et al. (1994) Ternds Genetics 10, 41-42.Schmied, M. et al. (1993) Am. J. Pathol. 143, 446-452.Wyllie, A. H. et al. (1980) Int. Rev. Cytol. 68, 251.Kerr, J. F. R. et al. (1972) Br. J. Cancer 26, 239-257.Duvall, E. & Wyllie, A. H. (1986) Immunol. Today 7, 115.Compton, M. M. (1992) Canc. Metastasis Rev.11, 105-119.

Allen, P. D., Bustin, S. A. & Newland, A. C. (1993) Blood Reviews 7, 63-73.

Cohen, J. J. & Duke, R. C. (1992) Annu. Rev. Immunol. 10, 267-293.Clarke, P. G. H. (1990) Anat. Embryol. 181, 195-213.

Johnson, E. M. & Deckwerth, T. L. (1993) Annu. Rev. Neurosci. 16, 31-46.

Batistatou, A. & Greene, L. A. (1993) J. Cell Biol. 122, 523-532.Strange, R. et al. (1992) Development 115, 49-58.

Carson, D. A. & Ribeiro, J. M. (1993) Lancet 341, 1251-1254.Edgington, S. M. (1993) Biotechnology 11, 787-792.

Gougeon. M.-L. & Montagnier, L. (1993) Science 260, 1269-1270.Hickman, J. A. (1992) Cancer Metastasis Rev. 11, 121-139.Afanasyev, V. N. et al. (1993) Cytometry 14, 603-609.

Bryson, G. J., Harmon, B. V. & Collins, R. J. (1994) Immunol. Cell Biology 72, 35-41.

Darzynkiewicz, Z. et al. (1992) Cytometry 13, 795-808.Ando, K. et al. (1994) J. Immunol. 152, 3245-3253.

Berges, R. R. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 10-14.

Gorczyca, W. et al. (1992) Int. J. Oncol. 1, 639-8.Gorczyca, W. et al. (1993) Exp. Cell Res. 207, 202-205.

Billig, H., Furuta, I. & Hsueh, A. J. W. (1994) Endocrinology 134, 245-252.

MacManus, J. P. et al. (1993) Neurosci. Lett. 1, -92.Mochizuki, H. et al. (1994) Neurosci. Lett. 170, 191-194.Oberhammer, F. et al. (1993) Hepatology 18, 1238-1246.

Portera-Cailliau, C. (1994) Proc. Natl. Acad. Sci. USA 91, 974-978.Preston, G. A. et al. (1994) Cancer Res. 54, 4214-4223.Weller, M. et al. (1994) Eur. J. Immunol. 24, 1293-1300.Zager, R.A. et al. (1994) J. Am. Soc. Nephrol. 4, 1588-1597.Cohen, G. M. et al. (1992) Biochem. J. 286, 331-334.Collins, R. J. et al. (1992) Int. J. Rad. Biol. 61, 451-453.Sei, Y. et al. (1994) Neurosci. Lett. 171, 179-182.Ansari, B. et al. (1993) J. Pathol. 170, 1-8.

Negoescu, A. et.al. (1998) Biochemica 3, 34-41

24www.roche-applied-science.com

5.3 Ordering Information

Apoptosis-specific physiological changeDNA fragmentation

Detection mode/ProductGel ElectrophoresisApoptotic DNA-Ladder KitIn situ assay

Pack sizeCat. No.

20 tests11 835 246 00111 684 795 91011 684 809 91011 684 817 910

In Situ Cell Death Detection Kit, Fluore-1 kit

scein (also usable for FACS)(50tests)In Situ Cell Death Detection Kit, APIn Situ Cell Death Detection Kit, POD

1 kit

(50 tests)1 kit (50 tests)70 tests (3.5 ml)70 tests (3.5 ml)2× 50 ␮l (20 tests)

Single reagents for TUNEL and supporting reagentsTUNEL APTUNEL PODTUNEL EnzymeTUNEL LabelELISA

Cell Death Detection ELISACell Death Detection ELISAPLUSCell Death Detection ELISAPLUS, 10×Cellular DNA Fragmentation ELISACell membrane alterations

Microscopy or FACSAnnexin-V-Alexa 568Annexin-V-BiotinAnnexin-V-FLUOSAnnexin-V-FLUOS Staining Kit

250 tests250 tests250 tests50 tests250 tests

03 703 126 00111 828 690 00111 828 681 00111 858 777 00111 988 549 001

1 kit1 kit (96 tests)1 kit

11 544 675 00111 774 425 00111 920 685 00111 772 457 00111 772 465 00111 767 305 001

3× 550 ␮l 11 767 291 001(30tests)

1 kit 11 585 045 001(500 tests)

25www.roche-applied-science.com

5.3 Ordering Information, continued

Apoptosis-specific physiological changeEnzymatic activity

Detection mode/ProductWestern Blot

Anti-Poly (ADP-Ribose) PolymeraseFIENA

Caspase 3 Activity AssayFluorimetric microplate AssayHomogenous Caspases Assay, fluorometricIn situ Assay

M30 CytoDEATH (formalin grade)M30 CytoDEATH, FluoresceinPack sizeCat. No.

100 ␮l1 kit100 tests1000 tests50 tests250 tests250 tests1 kit

11 835 238 00112 012 952 00103 005 372 00112 236 869 00112 140 322 00112 140 349 00112 156 857 00111 828 7 001

Expression of apopto-ELISAsis-related proteins

p53 pan ELISA

Single reagents

ProductDNase I recombinantPepsinTrypsin, solutionProteinase K, PCR gradePack Size 2 × 10,000 U1 g100 ml, sterile1250 ␮lCat. No.04 536 282 00110 108 057 00110 210 234 00111 9 3 001For further information please access our web-site address at:http://www.roche-applied-science.comor the Apoptosis special interest site:

http://www.roche-applied-science.com/apoptosisTriton is a Trademark of Rohm & Haas, Philadelphia, USA.

www.roche-applied-science.com26

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