tunel检测说明书

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Tunel说明书

For life science research only. Not for usein diagnostic procedures. FOR IN VITRO USE ONLY.In Situ Cell Death Detection Kit, TMR redVersion April 2006Kit 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. 12 156 792 910 1 Kit (50 tests) Store at Ϫ15 to Ϫ25°C1. PrefaceofContents1.1 Table (2)1. Preface1.1 Table of Contents 21.2 Kit Contents 32. Introduction (5)2.1Product Overview 5Information 82.2 Background3. Procedures and Required Materials (10)3.1Flow Chart 103.2Preparation of Sample Material 113.2.1Cell Suspension 113.2.2 Adherent Cells, Cell Smears, and Cytospin Preparations 123.2.3Tissue Sections 133.2.3.1 Treatment of Paraffin-Embedded Tissue 133.2.3.2 Treatment of Cryopreserved Tissue 153.3Labeling Protocol 163.3.1 Before you Begin 163.3.2 Labeling Protocol for Cell Suspensions 173.3.3Labeling Protocol for Adherent Cells, Cell Smears, Cytospin Preparations,and Tissues 183.3.4Labeling Protocol for Difficult Tissue 194. Typical results (20)5.Appendix (21)5.1Troubleshooting215.2 References 245.3 Ordering Information 251.2 KitContentsCaution 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, washimmediately with plenty of water. In case of accident or if you feelunwell 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 regulatedfor toxic waste.Kit Contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution•Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5×50␮l2 red Label Solution•Nucleotide mixture in reaction buffer•1× conc.• 5 × 550 ␮lAdditional Solutions Required 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.Procedure Equipment Reagents Preparation of sample material (section 3.2)•Cell suspension(section3.2.1)•Adherent cells, cell smearsand cytospin preparations (section 3.2.2.)•Cryopreserved tissue(section 3.2.3.2)•Shaker•V-bot-tomed 96-well micro-plate•Washing buffer: Phosphate bufferedsaline (PBS*)•Fixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared•Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.3.1)•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilledwater)•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 irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)•Micrococcal nuclease or •DNase I recombinant*•Cell suspensions (section3.3.2)•Adherent cells (section3.3.3)•Parafilm orcoverslips•HumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.4)•Plastic jar•Microwave•Humidifiedchamber•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 serum1.2 KitContents,continued2. Introduction2.1Product OverviewTest Principle 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 molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction. Arraybreaks 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 celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:•Detection of individual apoptotic cells in frozen and formalin fixedtissue 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 populationsby double staining procedures (6, 7).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test Interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand 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 withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained.To confirm apoptotic mode of cell death, the morphology of respectivecells should be examined very carefully. Morphological changes dur-ing apoptosis have a characteristic pattern. Therefore evaluation of cellmorphology is an important parameter in situations where there is anyambiguity 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)Assay Time1-2 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.Number of Tests The kit is designed for 50 tests.Kit Storage/ Stability 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.Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at single celllevel via fluorescence microscope and at cell pop-ulations via FACS analysis at very early stages (1,2, 6).Specific Preferential labeling of apoptosis versus necrosis(3, 4).Fast Short assay time (1–2 h).Convenient•No secondary detection system required.•One incubation and one washing step only.•Reagents are provided in stable, optimizedform.•No dilution steps required.•Application in combination with fluoresceinlabel possibleFlexible•Suitable for fixed cells and tissue. This allowsaccumulation, storage and transport of sam-ples (2, 5).•Double staining enables identification of typeand differentiation state of cells undergoingapoptosis (6).Function-tested Every lot is function-tested on apoptotic cells incomparison to a master lot.2.2 BackgroundInformationCell Death Two distinct modes of cell death, apoptosis and necrosis, can be dis-tinguished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic 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 tochromatin 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 describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant 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 antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).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 duringelectrophoresis. 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).2.2 BackgroundInformation,continued3. ProceduresandR equired M aterialsThe working procedure described below was published by R. Sgoncand colleagues (6). The main advantage of this kit is the use of tetra-methyl- rhodamine- dUTP to directly label DNA strand breaks with redfluorescence. This allows the direct detection of DNA fragmentationin the red channel and secondary labeling with fluorescein in thegreen channel by flow cytometry or fluorescence microscopy.3.1Flow ChartAssay Procedure The assay procedure is explained in the following flow chart.Cell suspensionAdherentcells, cellsmears, andcytospinprepara-tionsCryopre-served tissuesectionsParaffin-embeddedtissue sections↓↓↓↓FixationDewaxation Rehydration Protease treat-ment↓↓↓Permeabilisation↓Labeling reactionwith TUNEL reaction mixture↓↓Analyze byflow cyto-metry or byfluores-cencemicroscopyAnalyze by fluorescence microscopy3.2Preparation of Sample Material3.2.1Cell SuspensionPrelabeling For dual parameter flow cytometry with fluorescein-conjugated anti-bodies, incubate the cells prior to fixation with the cell surface marker.Additional Buff-ers and Equip-ment Required •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 microplateNote:Use of a V-bottomed 96-well microplate minimize cell loss dur-ing fixation, permeabilisation and labeling and allows simultaneous preparation of multiple samples.Procedure Please find in the following protocol the procedure for cell fixation and permeabilisation.Note: Fix and permeabilisate two additional cells for the negative andpositive labeling controls.Step Action1Wash test sample 3 times in PBS and adjust to 2 × 107cells/ml.2Transfer 100 ␮l/well cell suspension into a V-bottomed 96-wellmicroplate.3Add 100 ␮l/well of a freshly prepared Fixation solution to cellsuspension (final concentration 2% PFA).4Resuspend well and incubate 60 min at +15 to +25°C.Note:To avoid extensive clumping of cells, microplate should beincubated on a shaker during fixation.5Centrifuge microplate at 300 g for 10 min and remove fixative byflicking off or suction.6Wash cells once with 200 ␮l/well PBS.7Centrifuge microplate at 300 g for 10 min and remove PBS byflicking off or suction.8Resuspend cells in 100 ␮l/well Permeabilisation solution for2 min on ice (+2 to +8°C).9Proceed as described under 3.3.3.2.2 Adherent Cells, Cell Smears, and Cytospin PreparationsAdditional Solutions Required •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)Procedure The following table describes preparations of adherent cells, cellsmears and cytospin.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at +15 to +25°C2Rinse slides with PBS.3Incubate in Permeabilisation solution for 2 min on ice (+2to +8°C).4Proceed as described under 3.3.3.2.3Tissue Sections3.2.3.1 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).Additional Solutions Required •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 irradiationProcedure In the following table the pretreatment of paraffin-embedded tissuewith Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g., by heating at +60°C followed by washing inxylene and rehydration through a graded series of ethanol anddouble dist. water) (1, 33, 36).2Incubate tissue section for 15–30 min at +21 to +37°C withProteinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15–60 min at +37°C.3. Microwave irra-diation •Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH 6.0.•Apply 350 W microwave irradia-tion for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.3.1 Treatment of Paraffin-Embedded Tissue, continued3.2.3.2 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 preparedCryopreserved Tissue In the following table the pretreatment of Cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at +15 to +25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at Ϫ15 to Ϫ25°C.3Incubate slides in Permeabilisation solution for 2 min on ice (+2 to +8°C).4Proceed as described under 3.3.3.3Labeling Protocol 3.3.1Before you BeginPreparation of TUNEL Reaction MixtureOne 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.Additional Reagents Required •Micrococcal nuclease or •DNase I recombinant *ControlsTwo negative controls and a positive control should be included in each experimental set up.* available from Roche Applied ScienceStep Action1Remove 100 ␮l Label Solution (vial 2) for two negative con-trols.2Add 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.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 ␮l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control: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.3.3.2 Labeling Protocol for Cell SuspensionsAdditional Equipment and Reagents Required •Washing buffer: PBS •Humidified chamberProcedure Please refer to the following table.Step Action1Wash cells twice with PBS (200 ␮l/well).2Resuspend in 50 ␮l/well TUNEL reaction mixture.Note: For the negative control add 50 ␮l Label solution.3Add lid and incubate for 60 min at +37°C in a humidifiedatmosphere in the dark.4Wash samples twice in PBS.5Transfer cells in a tube to a final volume of 250-500 ␮l in PBS.6Samples 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).3.3.3Labeling Protocol for Adherent Cells, Cell Smears, Cytospin Preparations,and TissuesAdditional Equipment and Reagents Required •Washing buffer: PBS •Parafilm or coverslips •Humidified chamberProcedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add 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 mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Incubate slide in a humidified atmosphere for 60 min at +37°Cin the dark.5Rinse slide 3× with PBS.6Samples can directly be analysed under a fluorescence micro-scope or embedded with antifade prior to analysis. Use anexcitation wavelength in the range of 520-560 nm (maximum540 nm; green) and detection in the range of 570-620 nm(maximum 580 nm, red).3.3.4Labeling Protocol for Difficult TissueAdditional Equipment and Solutions Required •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•MicrowaveProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3•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!4Immerse 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% normalbovine serum.5•Rinse the slide(s) twice with PBS at +15 to +25°C.•Let excess fluid drain off.6Add 50 ␮l of TUNEL reaction mixture on the section.Note: For the negative control add 50 ␮l Label solution.7Incubate for 60 min at +37°C in a humidified atmosphere inthe dark.8•Rinse slide(s) three times in PBS for 5 min each.•Evaluate the section under a fluorescence microscope.results4. TypicalAssay Procedures•Incubate U937 cells at a density of 106 cells/ml in the presence ofcamptothecin (2 ␮g/ml, 4 h at +37°C) to induce apoptosis.•As control for non-apoptotic population, an aliquot of the cells isincubated 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)5.Appendix5.1TroubleshootingThis table describes various troubleshooting parameters.Problem Step/Reagent ofProcedurePossible Cause RecommendationNonspecific labeling Embedding oftissueUV-irradiation forpolymerization ofembedding material(e.g., methacrylate)leads to DNAstrand breaksTry different embedding material ordifferent polymerization reagent.Fixation Acidic fixatives(e.g., methacarn,Carnoy’s fixative)•Try 4% buffered paraformaldehyde.•Try formalin or glutaraldehyde.TUNELreactionTdT concentrationtoo highReduce concentration of TdT by dilu-ting it 1:2 up to 1:10 with TUNEL Dilu-tion Buffer*.Nucleases,PolymerasesSome tissues (e.g.,smooth muscles)show DNA strandbreaks very soonafter tissue prepa-ration.•Fix tissue immediately after organpreparation.•Perfuse fixative through liver vein.Some enzymes arestill active.Block with a solution containing ddUTPand dATP.High back-ground Sample Mycoplasma con-taminationMycoplasma Detection Kit*Highly proliferatingcellsDouble staining, e.g., with Annexin-V-Fluos*.Note: Measuring via microplate readernot possible because of too high back-ground.Fixation Formalin fixationleads to a yellowishstaining of cellscontaining melaninprecursors.Try methanol for fixation but take intoaccount that this might lead to reducedsensitivity.TUNEL reac-tionConcentration oflabeling mix is toohigh for mammacarcinoma.Reduce concentration of labeling mixto 50% by diluting with TUNEL DilutionBuffer.continued on next page* available from Roche Applied ScienceLow labeling Fixation Ethanol and metha-nol can lead to lowlabeling (nucleo-somes are notcross-linked withproteins during fix-ation and are lostduring the proce-dure steps)•Try 4% buffered paraformaldehyde.•Try formalin or glutaraldehyde.Extensive fixation leads to excessive cross-linking of proteins •Reduce fixation time.•Try 2% buffered paraformaldehyde.Permeabilisa-tion Permeabilisationtoo short so thatreagents can’treach their targetmolecules•Increase incubation time.•Incubate at higher temperature(e.g., +15 to +25°C).•Try Proteinase K (concentration andtime has to be optimized for eachtype of tissue).•Try 0.1 M sodium citrate at +70°Cfor 30 min.Paraffin-embedding Accessibility forreagents is too low•Treat tissue sections after dewaxingwith Proteinase K (concentration,time and temperature have to beoptimized 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 pageProblem Step/Reagent ofProcedurePossible Cause RecommendationNo signal on positive con-trol DNasetreatmentConcentration ofDNase is too low•For cryosections apply 3 U/mlDNase I recombinant.•For paraffin-embedded tissue sec-tions apply 1500 U/ml DNase Irecombinant.•In general, use 1 U/ml DNase Irecombinant, dissolved in 10 mMTris-HCl, pH 7.4 containing 10 mMNaCl, 5 mM MnCl2, 0.1 mM CaCl2,25 mM KCl and incubate 30 min at+37°C.•Alternative buffer: Tris- HCl pH 7.5containing 1 mM MgCl2 and 1 mg/ml BSA.Counter-staining diminishes TUNEL staining DNA stain Too high concentra-tions of DNA dyeUse 0.1–1 ␮g/ml BoBo–1 from Molec-ular Probes for counterstaining.Equivocal signals DoublestainingEarlier stage ofapoptosis thanstage detected byTUNEL reactionFor additional measurement of apopto-sis: M30 CytoDEATH* is suitable orAnnexin V – Fluos*.Problems with inter-pretation of results FACS Analysis Positive and nega-tive peaks are notdistinguishable,because too manyapoptotic bodiesacquired, apoptosisis too farChange apoptosis inducing procedure:2-3 Clusters should be visible in theFSC/SSC histogram:1.debris and apoptotic bodies2.whole cells3.shrinked cells gate should delete1.): clearly separated peaks.No signal for apop-tosisTime depends on cell line and inducingagents and should be optimized.Problem Step/Reagent ofProcedurePossible Cause Recommendation* available from Roche Applied Science。

TUNEL细胞凋亡检测试剂盒(显色法)说明书

TUNEL 细胞凋亡检测试剂盒(显色法)产品编号产品名称包装 C1098TUNEL 细胞凋亡检测试剂盒(显色法)50次产品简介：碧云天生产的显色法TUNEL 细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。

对于待检测的细胞或组织样品，经过生物素标记和后续的DAB 显色等步骤，即可在普通光学显微镜下观察到凋亡细胞。

细胞在发生凋亡时，会激活一些DNA 内切酶，这些内切酶会切断核小体间的基因组DNA 。

细胞凋亡时抽提DNA 进行电泳检测，可以发现180-200bp 的DNA ladder 。

基因组DNA 断裂时，暴露的3'-OH 可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase, TdT)的催化下加上生物素(Biotin)标记的dUTP(Biotin-dUTP)，随后和辣根过氧化物酶(HRP)标记的Streptavidin (Streptavidin-HRP)结合，最后在HRP 的催化下通过DAB 显色来显示凋亡细胞，从而可以通过普通光学显微镜检测到凋亡的细胞，这就是TUNEL(TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理。

本试剂盒有如下优点。

(1) 高灵敏度：背景染色极低，阳性染色强，可以在单细胞水平检测到细胞凋亡，同时由于凋亡早期就有DNA 断裂，可以检测到早期的细胞凋亡。

(2) 特异性好：TUNEL 检测时通常更容易标记凋亡细胞，而不容易标记坏死细胞。

(3) 快速：仅需约2-3个小时即可完成。

(4) 应用范围广：可以用于检测冷冻或石蜡切片中的细胞凋亡情况，也可以检测培养的贴壁细胞或悬浮细胞的凋亡情况。

(5) 实测效果好：参考图1。

图1. 本试剂盒的检测效果图。

A. HeLa 细胞未处理或用DNase I 室温孵育10分钟后的检测效果图。

自己翻译的罗氏tunel检测细胞凋亡试剂盒说明书

罗氏tunel检测细胞凋亡试剂盒说明书注意：Label溶液含有甲次砷酸盐和二氯化钴，严禁吸入和食入。

反应悬浮物收集于密闭、不易碎、有明确标识的容器中，按有毒废物处理。

除上表所列试剂外，还需准备以下溶液。

下表列出每步所需物品概览：特异性：TUNEL反应优先标记凋亡产生的DNA链断裂，从而辨别凋亡与坏死、以及由抑制细胞生长的药物或放射线产生的primary DNA链断裂实验干扰：假阴性：在某些型式的凋亡细胞中DNA链断裂可能缺失或不完全。

空间位阻，如细胞外元件可能阻止TdT到达DNA断裂处。

两种情况均能产生假阴性。

假阳性：在坏死晚期，可能产生大量的DNA片段DNA链断裂也可能在具有高增殖和代谢活动的细胞中出现。

两种情况均能产生假阳性。

为确认细胞死亡的凋亡型式，应认真进行每种细胞的形态学检查凋亡过程中产生的形态学改变尤其特征形式，因此，对于可以结果进行解释时，细胞形态评估是一项重要的参数样本：细胞离心涂片和细胞涂片在chamber slides上培养的黏附细胞冰冻或福尔马林固定、石蜡包埋样本分析时间：2-3小时，除外培养、固定和渗透检测次数：一个试剂盒50T试剂盒存储/稳定性：未开封试剂盒储存于-15～-25℃可稳定至标签上标明的效期。

1 流程图：2 样品准备2.1 黏附细胞、细胞涂片和细胞离心涂片需准备的其他试剂：Washing buffer：磷酸盐缓冲液（PBS）Blocking buffer封闭溶液：甲醇稀释的3% H2O2Fixation solution固定溶液：PBS配制的4%多聚甲醛，ph 7.4，新鲜配制Permeabilisation solution 渗透液：0.1%Triton1）X-100溶于0.1%柠檬酸钠溶液中，新鲜配制步骤：下表描述了细胞固定、内源性过氧化物酶封闭和细胞渗透过程。

2.2 组织部分2.2.1 福尔马林-包埋组织福尔马林包埋组织的预处理：可按4种不同的方式预处理。

翻译好的罗氏公司Tunel试剂盒操作说明书

罗氏 (Roche)公司 Tunel 试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL （TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA 的断裂情况。

其原理是荧光素（ fluorescein）标记的 dUTP 在脱氧核糖核苷酸末端转移酶（ TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂 DNA 的3’-OH 末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与 HRP 底物二氨基联苯胺（DAB ）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有 DNA 断裂，因而没有 3‘-OH 形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1 号（蓝盖） Enzyme Solution 酶溶液： TdT 10×、2号（紫盖） Label Solution 标记液：荧光素标记的 dUTP 1×、3号（棕瓶） Converter-POD:标记荧光素抗体的 HRP；自备试剂： PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB 工作液（临用前配制， 5 μl 20 ×DAB+1 μL 30%H2O2+94 μl PBS）、Proteinase K工作液（ 10-20 μg/ml in 10 mM Tris/HCl ，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于 0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、 DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl ，pH 7.5， 10 mM MgCl2 ，1 mg/ml BSA ）等。

凯基TUNEL细胞凋亡原位检测试剂盒

凯基TUNEL细胞凋亡原位检测试剂盒凯基TUNEL细胞凋亡原位检测试剂盒(通用)（BIOTIN标记POD法,适用于细胞、组织样本）使用说明书一、TUNEL制品说明凯基TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA 的断裂情况，其原理是生物素（biotin）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下,可以连接到凋亡细胞中断裂的DNA的3‘－OH末端，并可与连接了的辣根过氧化酶的链霉亲和素（Streptavidin-HRP）特异结合，在辣根过氧化酶底物二氨基联苯胺（DAB）的存在下，产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在普通显微镜下即可观察和计数凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）的凋亡原位检测。

本试剂盒特点●操作简便：使用Ready-to-Use型试剂，并配有Proteinase K 和DAB。

●高灵敏度：可以单一检出初期的凋亡细胞。

●高特异性：能特异性染色凋亡细胞。

●快速操作：整体操作约需3小时。

●用途广泛：可应用于组织切片、细胞样本等。

●方便观察：使用光学显微镜观察实验结果。

●高正确性：有阳性对照片的制备方法，可以确认试剂盒的有效性使用注意事项1．使用前请认真阅读本说明书，提前准备好相关试剂。

2．因本试剂盒中组分均为微量，使用前请离心集液。

3．为避免试验误差、降低试剂的损耗，建议使用精密度高的进口微量移液枪及枪头。

4． TdT 酶反应液最好在使用前根椐样本数量集中配制，再分别滴加于各样本片上，避免每个样本单独配制而产生的试剂损耗。

5. 为防止样本脱落，请使用硅烷（Silane）处理的载玻片或采用多聚赖氨酸铺片。

6. 固定好的样本可以在-20℃的70%乙醇中放置30分钟或至过夜，以改善细胞的渗透性。

翻译好的罗氏公司Tunel试剂盒操作说明书

罗氏(Roche)公司Tunel试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）Enzyme Solution 酶溶液：TdT 10×、2号（紫盖）Label Solution标记液：荧光素标记的dUTP 1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml–3 U/ml in 50 mM Tris-HCl，pH 7.5，10 mM MgCl2，1 mg/ml BSA）等。

罗氏荧光 tunel (11684795910)说明书

For life science research only. Not for use in diagnostic procedures.
In Situ Cell Death Detection Kit, Fluorescein
y Version 17
Content version: November 2012
Preface .....................................................................................................................................................2 Table of contents ................................................................................................................................................................. 2 Kit contents ............................................................................................................................................................................ 3 Introduction ............................................................................................................................................5 Product overview .................................................................................................................................................................. 5 Background information ................................................................................................................................................... 8 Procedures and required materials .................................................................................................10 Flow chart .............................................................................................................................................................................10 Preparation of sample material ....................................................................................................................................11 Cell suspension ..................................................................................................................................................................11 Adherent cells, cell smears, and cytospin preparations .....................................................................................12 Tissue sections ....................................................................................................................................................................12 Treatment of paraffin-embedded tissue ...................................................................................................................12 Treatment of cryopreserved tissue ..............................................................................................................................14 Labeling protocol ...............................................................................................................................................................15 Before you begin ................................................................................................................................................................15 Labeling protocol for cell suspensions ......................................................................................................................16 Labeling protocol for adherent cells, cell smears, cytospin preparations and tissues ...........................................................................................................................................................................17 Labeling protocol for difficult tissue ...........................................................................................................................18 Typical results ......................................................................................................................................19 Appendix ................................................................................................................................................20 Troubleshooting .................................................................................................................................................................20 References ............................................................................................................................................................................23 Related products ................................................................................................................................................................24

TUNEL细胞凋亡检测_荧光

TUNEL 细胞凋亡检测
1.二甲苯中脱蜡5-10分钟，换用新鲜的二甲苯，再脱蜡5-10分钟，无水乙醇5分钟，
90%乙醇2分钟，70%乙醇2分钟，蒸馏水2分钟。

2.滴加20μg/ml不含DNase的蛋白酶K（用pH7.4~8.0 10mM Tris），20-37℃作用15-30
分钟（不同组织的最佳作用温度和时间需自行摸索），用PBS洗涤3次（注意：这一步必须把蛋白酶K洗涤干净，否则会严重干扰后续的标记反应。

）。

3.标记液准备：从标记液中取100μl作为两个阴性对照，然后将TdT酶溶液（50μl）
加入到剩下的标记液（450μl）中，混匀。

4.样品的标记：在样品上加50μl 生物素标记液，37℃避光孵育60分钟（注意：孵
育时需注意在周围用浸足水的纸或药棉等保持湿润，以尽量减少标记液的蒸发）。

5.用PBS洗涤3次。

6.Mounting media 封片观察染色情况。

1.将细胞爬片在空气中风干，加入4% PFA 室温固定1h，PBS清洗3次。

2.破膜液（0.1% Triton X-100 in 0.1% 柠檬酸钠）4℃破膜2min，PBS清洗三次。

3.标记液准备：从标记液中取100μl作为两个阴性对照，然后将TdT酶溶液（50μl）
加入到剩下的标记液（450μl）中，混匀。

4.DNAase 处理阳性对照片，PBS洗涤三次。

5.样品的标记：在样品上加50μl 荧光标记液，37℃避光孵育60分钟（注意：孵育
时需注意在周围用浸足水的纸或药棉等保持湿润，以尽量减少标记液的蒸发）。

6.用PBS洗涤3次。

7.Mounting media 封片观察染色情况。

TUNEL细胞凋亡检测(荧光标记)操作流程

一、TUNEL实验试剂(1)DeadEnd™Fluorometric TUNEL System试剂盒购自Promega公司(产品编号G3250)(2)二甲苯购自国药集团化学试剂有限公司（产品编号80139118，CAS号108-38-3，500ml）(3)无水乙醇购自中国上海生工生物有限公司（产品编号ET0737-500ml）(4) 过氧化氢购自上海生工生物有限公司(产品编号H1976-500ml)(5) Proteinase K购自上海生工生物工程技术服务公司(产品编号BSP169-2ml)(6)多聚甲醛购自上海生工生物有限公司（产品编号PB0684-500g）(7) Propidium iodide购自Sigma（产品编号P4170-10MG）(8) SlowFade® Gold antifade reagent购自Life Technologies（产品编号36936）二、具体实验步骤A. 石蜡包埋组织预处理：1.二甲苯5min (500ml二甲苯)2.二甲苯5min（500ml二甲苯）3.无水乙醇5min（500ml无水乙醇）4.无水乙醇5min（500ml无水乙醇）5.80%乙醇5min（500x0.8=400ml无水乙醇）6.70%乙醇5min（500x0.7=350ml无水乙醇）7.PBS 5min（500ml）8.PBS 5min（500ml）9.PBS 5min（500ml）10.4%多聚甲醛in PBS，15min11.PBS 5min（500ml）12.PBS 5min（500ml）13.Proteinase K in PBS (100ul每个样品，共6ml), 8-10min14.PBS 5min（500ml）15.PBS 5min（500ml）16.4%多聚甲醛in PBS，5min17.PBS 5min18.PBS 5min共105分钟，实际约2个小时B. 凋亡检测1.每个样品，100ul平衡缓冲液覆盖细胞，室温5-10分钟2.在平衡细胞的同时，在冰上解冻核苷混合物，并且依照表1，准备足够量的用于所有实验的和可选阳性对照反应（见4.E节）的rTdT孵育缓冲液。

TUNEL SOP2012.9.24

TUNEL Standard Operating Procedure一、试剂盒来源美国罗氏（Roche）公司二、实验原理T UNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3’-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

图1 TUNEL 实验原理示意图PROTOCOL OF APOPTOSISPrinciple: The TUNEL (Terminal deoxynucleotidyl Transferase fluorescein-dUTP Nick End Labeling) method identifies apoptotic cells in situ by using terminal deoxynucleotidyl transferase (TdT) to transfer fluorescein-dUTP to the free 3'-OH of cleaved DNA. Then Detect the incorporated fluorescein with an anti-fluorescein antibody conjugating AP(alkaline phosphatase).Procedure:1. Paraffin embedding tissue sections2. Dewax, rehydrate sections by standard methods:Xylene and ethanol(absolute, 95%,90%,80,%70%,diluted in double distilled water)3. Optional: Inactivate endogenous POD/AP activity4. Wash slides with PBS(0.01M, PH7.2~7.4) 3 times, 3~5min/time5. Add protease and incubate (30 min, 37°C, protetase K, working solution:10~20ug/ml in 10mM Tris/HCl, PH7.4~8.0)6. Wash slides with PBS 3 times, 3~5min /time7. Permeabilize sections (2 min, on ice, permeabilisation solution0.1%TritonX-100, 0.1% sodium citrate, freshly prepared.8. Add TUNEL-reaction mixture and incubate (60 min, 37°C)9. Wash slides with PBS 3 times, 3~5min /time10. Optional: Analyze by fluorescence microscopy11. Add Anti-Fluorescein-AP or -POD and incubate (30 min, 37°C)12. Wash slides with PBS 3 times, 3~5min /time13. Add substrate and incubate (5–20 min, RT)14. Analyze by light-microscopy三、实验器材1. 光学器材（可选其一）：①正置光学显微镜及其成像系统（带荧光）②正置光学显微镜（Olympus）+数码相机（小镜头，易伸入显微镜头中拍照）2. 上行脱水缸：75%乙醇溶液、90%乙醇溶液、95%乙醇溶液、100%乙醇溶液、100%乙醇溶液、二甲苯（各1缸，共6个染色缸）3. 下行脱蜡与水化缸：二甲苯2缸（必要时3个）、100%乙醇溶液2缸、95%乙醇1缸、90%乙醇溶液1缸、75%乙醇溶液1缸、三蒸水1缸（8~9个）4．小型染色缸（PBS用）：3个5. 免疫组化笔或唇膏：1支6. 湿盒（带纱布）或自制湿盒：1~2个7. 盖玻片：若干片和若干规格（需处理后用）8. 吸管：若干支9. 加样器：200μL~1000μL、40μL~200μL、2μL~20μL10.枪头：200μL~1000μL、40μL~200μL、2μL~20μL11. 50.0mL螺口血清瓶（高压、消毒）：若干个12. 1.5 mL EP管、2.0 mL 与5.0 mL 冻存管（高压、消毒）：若干个13. EP管与冻存管架：若干个14. 苏木素染缸：1个15. 分色缸：1个16. PBS大缸：1个17.针式滤器（0.6μm）与10mL、50mL注射器：若干个18. 500mL容量瓶或盐水瓶：2个19. 有齿镊、小弯镊、小竹签（滴中性树胶用）：各1把（支）20. 清洁盖玻片储存盒：1个或清洁盖玻片储存缸1个21. 培养箱或温箱：37℃孵育用22. 烤箱：60℃考片用23.量筒：250mL、500mL、1000 mL、2000 mL，各1个24. 烧杯：50 mL、250 mL、500mL、1000 mL、2000 mL，各1个25. 玻棒：2支26. 磁力搅拌器：1个27. 50 mL 容量瓶1个28. 10mL试管（配DAB专用）若干支四、试剂1. 试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；2. 自备试剂（1）PBS（0.01M，PH 7.4）：中国福州迈新公司（2）单蒸水、三蒸水或超纯水（3）二甲苯（4）梯度乙醇溶液（100%、95%、90%、75%）：采用95%与无水乙醇配制（5）DAB显色试剂盒（含20×DAB、30%H2O2、PBS）：中国福州迈新公司（6）蛋白酶K（Proteinase K）：（MERCK公司）①母液（1mg/mL）： -20℃保存；②工作液：10-20 μg/mL in 10 mM Tris/HCL，pH 7.4-8，4℃保存，以1月内使用为宜。

翻译好的罗氏公司Tunel试剂盒操作说明方案

罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。

TUNEL说明书

TUNEL说明书1 介绍TUNEL是提供单细胞水平细胞凋亡的稳定系统，能够迅速、快捷、精确的检测出凋亡细胞。

该试剂盒可以通过测定核DNA片段检测组织切片和培养细胞的凋亡细胞。

多数高等真核生物的细胞都通过启动自身的细胞自杀程序实现程序性死亡或细胞凋亡。

凋亡在发育、内环境稳定和一些疾病中具有重要作用。

凋亡具有某些特定的形态学特征，包括细胞膜起泡，细胞核和细胞质固缩，染色质浓缩，并且不发生局部炎症反应。

细胞死亡与此相反，它的特点是细胞肿胀，染色质絮凝，细胞膜完整性破坏，细胞溶解和产生及局部炎症反应。

凋亡过程中，内源性Ca2+、Mg2+依赖性核酸内切酶被激活，DNA被降解而形成末端为3’－OH、含180～200碱基对的不同倍数的核苷酸片段。

TUNEL 可用于多种细胞凋亡的检测，已经经过验证的应用范围: Vibratome® 神经组织切片, Jurkat 细胞, HL-60细胞这本技术小册子包括检测组织切片和茴香霉素诱导的HL-60细胞的细胞凋亡。

检测原理: DeadEnd™ Colorimetric TUNEL 系统使用改良的TUNEL (TdT-mediated dUTP Nick-End Labeling)对凋亡细胞的断裂DNA进行末端标记。

使用末端脱氧核苷转移酶(TdT)将生物素标记的核苷被掺入到DNA的3′-OH末端。

然后，辣根过氧化物酶标记的链霉亲和素(Streptavidin HRP)结合在上述生物素标记的核苷上，可以通过过氧化物酶的底物——过氧化氢和稳定的显色剂氨基联苯胺(DAB)检测到。

用这种程序，凋亡细胞的核被染成深棕色。

2 产品内容G7361平衡缓冲液（G327B）——4.8ml末端脱氧核苷酰酶酸转移酶（M828B）——20ul抗生物素蛋白链菌素辣根过氧化物酶（G714A）——40ul生物素化的核苷混合物（G715A）——20ul蛋白酶K（V302A）——10mgG7362塑料盖玻片（G326B）——2020X SSC（G329B）——20mlDAB 20X 色原体（G716A）——200ul20X DAB底物缓冲液（G717A）——200ul20X过氧化氢（G718A）——200ul储存条件: 将平衡缓冲液, TdT酶, 生物素标记的核苷混合物和蛋白酶K 储存于–20°C。

翻译好的罗氏公司Tunel试剂盒操作说明方案

翻译好的罗氏公司T u n e l试剂盒操作说明方案集团档案编码：[YTTR-YTPT28-YTNTL98-UYTYNN08]罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。

罗氏TUNEL使用说明书(中文)

罗氏公司TUNEL细胞凋亡检测程序（In situ cell death detection kit-POD法）一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3'-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70％）、DAB工作液（临用前配制，5 µl 20×DAB＋1μL30％H2O2＋94 µl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl, pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5, 10 mM MgCl2，1 mg/ml BSA）等。

TUNEL说明书

不适用于诊断，仅供生命科学实验使用。

仅供体外使用。

TUNEL细胞凋亡原位检测试剂盒（标记POD）本试剂盒可标记DNA链末端，即TUNEL技术。

用于单个细胞水平凋亡（细胞程序性死亡）的免疫组织化学检测和定量分析。

光镜检测。

批号：11684817910一盒可用50次储存：-15—-25℃操作指南2006.01.1.2 试剂盒组成注意事项本品标记溶液含有二甲基砷酸盐，容易被吸入而产生毒性和导致呕吐；也含有二氯化钴，吸入后容易致癌。

因此应避免接触并遵循相关的操作说明。

使用过程中不能吃、喝和抽烟。

如果接触到皮肤，应立即用大量水冲洗干净。

如果感觉不适或者突发其它情况，应立即就医。

酶标反应应在致密的、无损坏的容器中进行，收集上清液后应标明成分。

垃圾应作为有毒废物进行处理。

注意：与先前的试剂盒/管不同，次试剂盒的酶溶解液不再含有毒的二甲砷基酸盐，因此瓶1没有毒性。

试剂盒组成请参照下表对比试剂盒组成试剂盒外自备仪器和试剂出上表所列试剂外，实验者需制备系列溶液。

下表列出了在不同实验步骤中所需要的试剂。

在每步操作前都给出了详细的说明。

2 引言2.1 产品描述实验原理在细胞凋亡过程中，基因组DNA 会断裂产生双链、低分子量的DNA 片段和高分子量的单链DNA 断端（缺口），这些DNA 链缺口可以利用酶标记核苷酸3’末端方法来识别。

应用原位细胞凋亡检测试剂盒具有准确、快速、简单、非辐射等特点，可以用来对细胞或者组织中的单个细胞进行检测并定量，因而次法被用在很多分析系统中，例如：●在基础研究和日常病理中检测冰冻和福尔马林固定的组织切片。

●在肿瘤研究和临床癌基因研究中确定某些恶性肿瘤对某种药物的敏感性。

●通过双染色操作，确定经历死亡的异常增生细胞的分型。

专一性TUNEL反应可以更好的标记通过凋亡产生的DNA链末端，因此就可以区分出凋亡与坏死，以及由抗肿瘤药物或者放射诱发的初始DNA链末端。

试验干扰假阴性：在某些形式的细胞凋亡中，DNA逃逸酶切或者不完全（37）。

TUNEL 绿色荧光检测试剂盒说明书

4.2. DNA 酶处理阳性对照的步骤（可选的）............................................................................... 8
4.3. 标记及检测 .......................................................................................................................... 9
4. 4. 流式细胞术检测悬浮细胞的步骤 ..................................................................................... 11
五、操作实例：喜树碱（Camptothecin）诱导的 HL-60 细胞凋亡的检测 ................................ 12
TUNEL 绿色荧光检测试剂盒 M3026
用户手册
TUNEL 绿色荧光检测试剂盒
Fluorescence and Colorimetric TUNEL Apoptosis Assay Kit
用户手册
产品编号：M3026 M3026-1 50 次
上海美季生物技术有限公司
上海市中山南二路 777 号 2 号搂 2303
上海美季生物技术有限公司
上海市中山南二路 777 号 2 号搂 2303

Mbchem™品牌为上海美季生物技术有限公司所有电话：86-021-51873168 传真:86-021-51875086-800
本 use only. Not for human or diagnostic use.

罗氏TUNEL使用说明书中文

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3'-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

tunel检测说明书

一、TUNEL试剂盒说明凯基一步法TUNEL细胞凋亡检测试剂盒(One Step TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法，可检测细胞在凋亡过程中细胞核DNA的断裂情况，其原理是绿色荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下,可以连接到凋亡细胞中断裂的DNA的3'－OH末端，可用荧光显微镜检测。

由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被标记。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）的凋亡原位检测。

对于经过固定和洗涤的细胞或组织，只要经过一步染色反应，洗涤后就可以通过荧光显微镜检测到凋亡细胞。

本试剂盒特点●操作简便：使用Ready-to-Use型试剂，只需一步染色反应，洗涤后即可观察。

●高灵敏度：可以单一检出初期的凋亡细胞。

●高特异性：能特异性染色凋亡细胞。

●快速操作：仅需约1-2个小时即可完成整体操作。

●方便观察：可使用荧光显微镜观察实验结果。

●高正确性：有阳性对照片的制备方法，可以确认试剂盒的有效性●应用范围广：可以用于冷冻或石蜡切片中的组织细胞凋亡检测，也可检测培养的贴壁细胞或悬浮细胞的凋亡情况。

二、TUNEL试剂盒组分注意事项1．使用前请认真阅读本说明书，提前准备好相关试剂。

2．因本试剂盒中组分均为微量，使用前请离心集液。

3．为避免试验误差、降低试剂的损耗，建议使用精密度高的进口微量移液枪及枪头。

4． TdT 酶反应液最好在使用前根椐样本数量集中配制，再分别滴加于各样本片上，避免每个样本单独配制而产生的试剂损耗。

5. 用户自备：二甲苯、多聚甲醛、PBS、H2O2、TritonX-100、柠檬酸钠、盖玻片、载玻片、染色缸。

三、操作流程概览细胞涂片、贴壁细胞爬片等细胞样本四、检测样本的预处理TUNEL检测时样本的预处理是试验的关键所在，本说明书推荐的条件仅为普遍情况，用户需根椐自已的样本材料及首次试验结果来调整各个条件（参照Page 10），如处理时间、处理浓度等，来优化出适合自身样本的试验条件，从而做出客观的试验结果。

翻译好的罗氏公司Tunel试剂盒操作说明书

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

tunel检测说明书

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