BD ApoAlert DNA Fragmentation Assay Kit
Contents
1. Rev Cytol 68 251 306 Zhang C Ao Z Seth A amp Schlossman S F 1996 A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells J Immunol 157 3980 3987 Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 19 BD ApoAlert DNA Fragmentation Assay Kit User Manual X Related Products For a complete listing of all BD Biosciences Clontech products please go to www bdbiosciences com clontech Products Apoptosis Inducing Reagents Human TNF a Apoptosis Inhibiting Reagents BD ApoAlert Caspase 3 Inhibitor DEVD CHO BD ApoAlert Caspase 3 Inhibitor DEVD fmk BD ApoAlert Caspase 8 Inhibitor IETD fmk e BD ApoAlert Caspase 1 Inhibitor YVAD cmk e BD ApoAlert Caspase Inhibitor VAD fmk Apoptosis Assay Kits BD ApoAlert Caspase 3 Assay Kits Fluorescent Colorimetric BD ApoAlert Caspase 8 Assay Kits Fluorescent Colorimetric BD ApoAlert Annexin V FITC Apoptosis Kit BD ApoAlert Annexin V EGFP Apoptosis Kit e BD ApoAlert Mitochondrial Membrane Sensor Kit e BD ApoAlert Cell Fractionation Kit Apoptosis Assay Plates BD ApoAlert Caspase 3 Assay Plate BD ApoAlert Caspase Profiling Plate Apoptosis Detection Vectors e BD ApoAlert pCaspase3 Sensor Vector BD ApoAlert pDsRed2 Bid Vector e BD ApoAlert pd4EGFP Bid Vector BD Bioscie2. kb fragments prior to or in the absence of internucleosomal fragmentation EMBO J 12 3679 3684 Piqueras B Autran B Debre P amp Gorochov G 1996 Detection of apoptosis at the single cell level by direct incorporation of fluorescein dUTP in DNA strand breaks Bio Techniques 20 634 640 Roy C Brown D L Little J E Valentine B K Walker P R Sikorska M Leblanc J amp Chaly N 1992 The topoisomerase Il inhibitor teniposide VM 26 induces apoptosis in unstimulated mature murine lymphocytes Exp Cell Res 200 416 424 Schwartzman R A amp Cidlowski J A 1993 Apoptosis the biochemistry and molecular biology of programmed cell death Endocrine Rev 14 133 151 Thompson C B 1995 Apoptosis in the pathogenesis and treatment of disease Science 267 1456 1462 Thornberry N A amp Littlewood Y 1998 Caspases Enemies Within Science 281 1312 1316 Walker P R Smith C Youdale T Leblanc J Whitfield J F amp Sikorska M 1991 Topoisomerase ll reactive chemotherapeutic drugs induce apoptosis in thymocytes Cancer Res 51 1078 1085 Wijsman J H Jonker R R Keijzer R Van de Velde C J H Cornelisse C J amp Van Dierendonck J H 1993 A new method to detect apoptosis in paraffin sections In situ end labeling of fragmented DNA J Histochem Cytochem 41 7 12 Wyllie A H Kerr J F R amp Currie A R 1980 Cell death The significance of apoptosis ntl
3. 0 100 ul of the cell suspension onto a poly L lysine coated or silanized slide GENTLY spread the cell suspension with a clean slide Fix cells by immersing the slides in a Coplin jar containing fresh 4 formaldehyde PBS at 4 C for 25 min Note You may store fixed cells for up to two weeks at 209C in 70 ethanol Immerse the slides in a Coplin jar containing fresh PBS for 5 min at room temperature Repeat PBS wash 12 Permeabilize the cells by immersing the slides in a Coplin jar containing prechilled 0 2 Triton X 100 PBS Incubate 5 min on ice Optional Prepare a DNase treated positive control a Add 100 ul of DNase I Buffer Incubate at room temperature for 5 min Gently tap the slide to remove the liquid Add 100 ul of DNase I Buffer containing 0 5 1 ug ml DNase Incubate at room temperature for 10 min Gently tap the slide to remove the liquid Immerse 3 4 times in H5O in a Coplin jar reserved for DNase treated samples Immerse the slides in a Coplin jar containing fresh PBS for 5 min at room temperature Transfer the slides to a second Coplin jar containing fresh PBS for 5 min at room temperature Proceed to Section VI OQ aa T o O Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 11 BD ApoAlert DNA Fragmentation Assay Kit User Manual V Sample Preparation for Microscopic Detection continued C Tissue Sections This pr
4. BD ApoAlert DNA Fragmentation Assay Kit User Manual Cat Nos 630107 630108 PT3137 1 PR4Y988 Published 17 November 2004 BD ApoAlert DNA Fragmentation Assay Kit User Manual Table of Contents Il Introduction 3 ll List of Components 6 Ill Additional Materials Required 7 IV Controls 9 V Sample Preparation for Microscopic Detection 10 A Adherent Cells 10 B Suspended Cell Cultures 11 C Tissue Sections 12 VI Detection by Microscopy 14 VII Detection by Flow Cytometry 16 Vill Troubleshooting Guide 18 IX References 19 X Related Products 20 Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use BD Biosciences Clontech products may not be resold modified for resale or used to manufacture commercial products without written approval of BD Biosciences Clontech Triton is a trademark of The Dow Chemical Company BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2004 BD BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 2 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual Introduction Apoptosis or programmed cell death plays a fundamental role in many normal biological processes as well as in several disease states Wyllie etal 1980 Ellis et al 1991 Cohen et al 1992 Thompson 1995 Nic
5. M NaCl 300 mM NagCitrate H O If stored at 20 C 20X SSC must be warmed to room temperature to ensure that all salts are in solution prior to 10X dilution with deionized H O After opening the kit 20X SSC can be stored at room temperature BD Biosciences Clontech www bdbiosciences com 6 Protocol No PT3137 1 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual lll Additional Materials Required For working with cell cultures Sections V A amp V B Poly L lysine coated microscope slides Apply 50 100 ul of a 0 1 w v aqueous solution of poly L lysine Sigma Cat No P8920 to the surface of sterile glass microscope slide Spread evenly over the portion of the slide that will be used for growing and fixing cells Air dry the slides for 1 hr and store at 4 C for up to one week Alternatively use Lab Tek Chamber Slides or equivalent commercial slides Coplin jars filled with Phosphate buffered saline PBS pH 7 4 6 jars 4 Formaldehyde PBS pH 7 4 1 jar Prepare formaldehyde PBS solutions immediately before use Combine 70 ml of PBS and 25 ml of 16 methanol free formaldehyde Polyscience Inc Cat No 18814 or Aldrich Cat No 44124 4 Adjust the pH to 7 4 with 1N NaOH 0 2 Triton X 100 PBS 1 jar Deionized H O 1 jar reserve for DNase treated samples DNase I Buffer 40 mM _ Tris HCl pH 7 9 10 mM _ NaCl 6 mM MgCl 10 mM CaCl DNase I 0 5 ug to 1 ug ml final concentr
6. aldehyde PBS pH 7 4 Prepare formaldehyde PBS solutions immediately before use Combine 90 ml of PBS and 6 25 ml of 16 methanol free formaldehyde Adjust the pH to 7 4 with 1 N NaOH 20 mM EDTA pH 8 0 0 1 Triton X 100 BSA PBS Combine 5 ml PBS 5 ml 0 2 Triton X 100 50 mg BSA Propidium iodide PBS PI PBS 1 jar Dissolve 10 mg of propidium iodide Sigma Cat No P4170 in 10 ml of PBS Store in the dark at 4 C PI RNase PBS 2 5 ug ml Propidium iodide 0 5 ug ml DNase free RNase BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 8 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual IV Controls We recommend including positive and negative controls There are two types of positive controls Biological controls Use an apoptosis inducing reagent to induce apopto sis Table describes the modes of action of several such reagents some are available from BD Biosciences Clontech see Related Products TABLE Ii MODES OF ACTION OF APOPTOSIS INDUCING REAGENTS Agent Mode of Action Actinomycin D Inhibits RNA synthesis Anti Fas antibody Binds and activates Fas cell surface protein CD95 clone CH 11 Cycloheximide Inhibits protein synthesis C2 Ceramide Second messenger in sphingomyelin pathway Dexamethasone _ Binds glucocorticoid receptor Etoposide Inhibits topoisomerase Human TNF a Binds TNF receptor when applied with cyclohexamide Staurosporine Inhibit
7. ashing by washing the slides 3 x 5 min with Triton X 100 BSA PBS No positive signal This problem usually indicates poor permeabilization with Triton X 100 Steps V A 7 V B 12 or VII 16 or with Proteinase K Step V C 11 Address the problem by incubating longer with the permeabilization reagent Tissue sections are loose Tissue slides can loosen for two reasons 1 poor preparation of the slides prior to mounting of tissue sections or 2 overdigestion by Proteinase K Step V C 11 Correct the first problem by switching from poly L lysine coated slides to slides coated with 3 aminopropyl triethoxysilane Ben Sasson ef al 1995 Correct the second problem by optimizing the Proteinase K incubation time Too few cells for analysis You can lose cells at several points If this problem occurs consistently first increase the starting number of cells If you are preparing suspension cells for attachment to slides Section V B try washing the cells with PBS 1 BSA during the centrifugation steps If a cytospin centrifuge is available use it to prepare your samples Finally try washing the cells with PBS 1 BSA during the centrifugation steps in Section VII BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 18 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual IX References Ben Sasson S A Sherman Y amp Gavrieli Y 1995 Identification of dying cells by in sit
8. ation Suspension cells only Refrigerated centrifuge for collecting cells For working with paraffin embedded tissue sections Section V C Coplin jars filled with Xylene 2 jars Ethanol rehydration series 100 ethanol 2 jars 95 ethanol 1 jar 85 ethanol 1 jar 70 ethanol 1 jar 50 ethanol 1 jar 0 85 NaCl 1 jar PBS 7 jars 4 Formaldehyde PBS pH 7 4 2 jars 20 ug ml Proteinase K solution 1 jar Combine 2 ul of 10 mg ml Proteinase K provided in the kit and 998 ul of 100 mM Tris HCl pH 8 0 50 mM EDTA Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 7 BD ApoAlert DNA Fragmentation Assay Kit User Manual lll Additional Materials Reguired continued For microscopic detection of apoptosis Section VI Forceps for handling slides and coverslips 37 C incubator dark humidified 2X SSC Dilute 20X SSC included in the kit 10 fold with HO Coplin jars filled with e PBS 3 jars e Propidium iodide PBS PI PBS 1 jar 10 ug ml Final Concentration Stock Dissolve 10 mg of propidium iodide Sigma Cat No P4170 in 10 ml of PBS Store in the dark at 4 C e Deionized H O 3 jars PI RNase PBS 10 ug ml Propidium iodide 0 5 ug ml DNase free RNase Anti Fade reagent Molecular Probes Cat No 87461 For detection of apoptosis by flow cytometry Section VII Refrigerated centrifuge for collecting cells 37 C water bath PBS 1 Form
9. cells by incubating on ice with 5 ml of prechilled 0 2 Triton X 100 Collect the cells by centrifugation at 300 x g for 10 min at 4 C Remove supernatant and gently resuspend the cells in 5 ml of PBS Collect the cells by centrifugation at 300 x g for 10 min at 4 C Remove the supernatant and gently resuspend the cells in 1 mlof PBS Transfer 2 x 10 cells to an amber 1 5 ml microcentrifuge tube Note Protect samples from light from this point Collect the cells by centrifugation at 300 x g for 10 min at 4 C Carefully aspirate the PBS Gently resuspend the cells in 80 ul of Equilibration Buffer Incubate at room temperature for 5 min BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 16 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual VII Detection by Flow Cytometry continued 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Thaw the Nucleotide Mix on ice and prepare TdT incubation buffer for all experimental samples positive controls and biological negative controls Total No of Total Component Volume Reactions Volume Equilibration Buffer 45 ul a o yl Nucleotide Mix 5 ul 5 ul TdT Enzyme 1 ul r gt ul 51 ul A ul For your TdT minus negative control prepare TdT minus control incubation buffer by replacing the TdT Enzyme with deionized HO Note Protect Nucleotide Mix and TdT incubation buffer from light Keep
10. holson 1996 Apoptotic cells undergo many distinct morphological and biochemical changes including fragmentation of nuclear DNA Schwartzman amp Cidlowski 1993 Walker et al 1991 Oberhammer et al 1993 Roy et al 1992 During apoptosis cellular endonucleases cleave nuclear DNA between nucleosomes producing a mixture of DNA fragments whose lengths vary in multiples of 180 to 200 bp The BD ApoAlert M DNA Fragmentation Assay Kit detects apoptosis induced nuclear DNA fragmentation via a fluorescence assay The assay is based onterminal deoxynucleotidyl transferase TdT mediated dUTP nick end labeling TUNEL Gavrieli et al 1992 Piqueras et al 1996 Facchinetti et al 1991 Wijsman et al 1993 TdT catalyzes incorporation of fluorescein dUTP at the free 3 hydroxyl ends of fragmented DNA fluorescein labeled DNA can be detected via fluorescence microscopy or flow cytometry This kituses a direct labeling procedure for detecting DNA fragmentation at the single cell level With a fluorescence microscope equipped with FITC filters you can visualize areas of apoptotic cells in situ in tissue sections The kit can be used with adherent cells and cells in suspension Other BD ApoAlert Apoptosis Detection Kits Although the TUNEL assay is widely used for detecting apoptosis DNA fragmen tation occurs relatively late in the process Recent studies have elucidated some molecular events that are characteristic of the early stages of apopt
11. ic cells Bid resides in the cytosol as soluble protein Upon induction of apotosis Bid trafficks to the mitochondria In cells expressing the Bid d4EGFP fusion Bid trafficking can be detected by fluorescence microscopy BD Biosciences Clontech also offers apoptosis inducing reagents as well as antibodies protease inhibitors stains and other reagents for apoptosis research see Related Products Section IX Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 5 BD ApoAlert DNA Fragmentation Assay Kit User Manual ll List of Components Upon receipt store the kit at 20 C After first use store 20X SSC and plastic coverslips at room temperature Store all other components at 20 C Important The nucleotide mix is sensitive to light Cat No 630107 25 assays Eguilibration Buffer 4x1 ml Nucleotide Mix 2x 62 5 ul TdT Terminal Transferase 2x 312 5 units 20X SSC see note below 29 ml e Proteinase K 10 mg ml 440 ul e Plastic Coverslips 25 Composition of components e Equilibration Buffer Cat No 630108 100 assays 10x 1 6 ml 8x62 5 ul 5 x 500 units 116 ml 1 85 ml 100 200 mM Potassium cacodylate pH 6 6 at 25 C 25 mM Tris HCI pH 6 6 at 25 C 0 2 mM DTT 0 25 mg ml BSA 2 5 mM Cobalt chloride Potassium cacodylate dimethylarsinic acid is a mild irritant Avoid skin and eye contact by wearing gloves and safety glasses 20X SSC 3
12. ic substrates for four different caspases immobilized in separate wells When cell lysate containing the active caspase of interest is added to the wells the caspase cleaves the substrate yielding a fluorescent product that can be detected with a standard fluorescence microplate reader The BD ApoAlert pCaspase3 Sensor Vector Cat No 630222 encodes a fusion of BD Living Colors Enhanced Yellow Fluorescent Protein EYFP and the nuclear localization signal NLS of SV40 T antigen It can be used to detect the onset of caspase 3 activity in mammalian cells with fluorescence microscopy BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 4 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual Introduction continued The BD ApoAlert pDsRed2 Bid Vector Cat No 632419 encodes a fusion of a BD Living Colors Fluorescent Protein DsRed2 a red fluorescent protein and Bid a member of the Bcl 2 family In healthy non apototic cells Bid resides in the cytosol as soluble protein Upon induction of apotosis Bid trafficks to the mitochondria In cells expressing the Bid DsRed fusion Bid trafficking can be detected by fluorescence microscopy The BD ApoAlert pd4EGFP Bid Vector Cat No 630116 encodes a fusion ofa BD Living Colors Fluorescent Protein dEGFP a destablilized enhanced green fluorescent protein and Bid a member of the Bcl 2 family In healthy non apotot
13. iling reaction place the slides in a dark humidified 37 C incubator for 60 min Note Ensure high humidity by placing wet paper towels in the bottom of dry incubators Using forceps remove the plastic coverslips Terminate the tailing reaction by immersing the slides in a Coplin jar containing 2X SSC Incubate at room temperature for 15 min BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 14 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual VI Detection by Microscopy continued 15 Wash the cells by transferring the slides to a fresh Coplin jar filled with PBS Incubate at room temperature for 5 min 16 Repeat Step 15 twice 17 Stain the cells with propidium iodide Pl by incubating the slides in a Coplin jar containing 40 ml of fresh PI PBS at room temperature for 5 10 min Note If PI staining is too intense include 0 5 ug ml of DNase free RNase at this step 18 Wash the cells by transferring the slides to a fresh Coplin jar filled with deionized H O and incubate at room temperature for 5 min 19 Repeat Step 18 twice 20 Optional Add a drop of Anti Fade reagent and cover the treated portion of the slide with a glass coverslip 21 Optional Seal the edges of the coverslip with rubber cement or clear nail polish and allow to dry for at least 5 10 min You may store sealed slides overnight at 4 C in the dark View the slides as soon as possible Apoptotic cells
14. llow the liquid to drain thoroughly and place the slides on a flat surface Prepare 20 ug ml Proteinase K solution Section III and cover each section with 100 ul of it Incubate at room temperature for 5 min Note Optimizing the length of Proteinase K treatment may be useful For example sections thicker than 5 um may require longer treatments However overtreatment may loosen sections causing them to wash off the slides during subsequent steps Immerse the slides in a Coplin jar containing PBS and incubate at room temperature for 5 min BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 12 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual V Sample Preparation for Microscopic Detection continued 13 Transfer the slides to a Coplin jar containing 4 formaldehyde PBS and incubate at room temperature for 5 min 14 Wash the slides by immersion in a Coplin jar containing PBS and incubate at room temperature for 5 min 15 Proceed to Section VI Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual VI Detection by Microscopy This assay procedure can be used for any sample obtained in Section V See Section III for information about Coplin jars and solutions Separate Coplin jars that have been used with DNase from DNase free Coplin jars 1 2 3 10 Remove the slides fro
15. m PBS and tap gently to remove excess liquid Cover the cells in 100 ul of eguilibration buffer Using forceps gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid Eguilibrate at room temperature for 10 min Thaw Nucleotide Mix on ice and prepare TdT incubation buffer for all experimental samples positive controls and biological negative controls Total No of Total Component Volume Reactions Volume Equilibration Buffer 45 ul u Nucleotide Mix 5 ul ul TdT Enzyme 1 ul ul 51 ul ul For your TdT minus negative control prepare TdT minus control incubation buffer by replacing the TdT Enzyme with deionized HO Note Protect Nucleotide Mix and TdT incubation buffer from light Keep on ice at all times Using forceps remove the plastic coverslip and gently tap the slides to remove excess liquid Carefully blot dry around the edges with tissue paper Gently place 50 ul of TdT incubation buffer on the cells on a 5 cm area Note Do not allow the cells to dry out Optional If performing a TdT minus negative control add 50 ul of TdT minus control incubation buffer From this point protect samples from light by wrapping Coplin jars in aluminum foil or keeping the jars in a covered box 11 12 13 14 Using forceps gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid To perform the ta
16. nces Clontech www bdbiosciences com 20 Cat No 630203 630204 630207 630209 630205 630208 630214 630215 630216 630217 630218 630219 630220 630221 630109 630110 630113 630114 630106 630105 630223 630224 630225 630226 630222 632419 630116 Protocol No PT3137 1 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual Notes Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 21 BD ApoAlert DNA Fragmentation Assay Kit User Manual Notes BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual Notes Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 23
17. on ice at all times Collect the cells by centrifugation at 300 x g for 10 min at 4 C Carefully aspirate the supernatant Resuspend in 50 ul of TdT incubation buffer Incubate at 37 C in a water bath for 60 min protected from direct light Mix the cell suspension by gently pipetting every 15 min Add 1 ml of 20 mM EDTA to terminate the reaction and mix by gentle vortexing Collect the cells by centrifugation at 300 x g for 10 min at 4 C Carefully aspirate the supernatant Resuspend the cells in 1 ml of 0 1 Triton X 100 BSA PBS Repeat Steps 33 35 Collect the cells by centrifugation at 300 x g for 10 min at 4 C Carefully aspirate the supernatant Stain the cells with propidium iodide Pl by gently resuspending them in 0 5 ml of PI RNase PBS Incubate at room temperature in the dark for 15 30 min Analyze the cells as soon as possible Apoptotic cells can be collected based on green fluorescence at 520 20 nm All cells can be sorted based on red fluorescence at gt 620 nm Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 17 BD ApoAlert DNA Fragmentation Assay Kit User Manual VIII Analysis of Results amp Troubleshooting Guide e High background High background can be caused by nonspecific incorporation of fluores cein 12 dUTP This problem can occur if the cells dry out at Step VI 11 or if they are insufficiently washed at Step VI 17 Correct for insufficient w
18. osis Zhang et al 1996 Koester et al 1997 Green 1998 Nicholson amp Thornberry 1997 Thornberry amp Littlewood 1998 As a result the TUNEL assay can be comple mented with other assays that detect apoptosis at earlier stages The BD ApoAlert Caspase 3 Fluorescent Assay Kits Cat Nos 630214 amp 630215 and BD ApoAlert Caspase 3 Colorimetric Assay Kits Cat Nos 630216 amp 630217 provide assays for caspase 3 protease activity or other proteases that recognize the caspase 3 cleavage se quence DEVD Induction of caspase 3 activity is one of the earliest events in Fas receptor induced apoptosis however caspase 3 activity is induced somewhat later in staurosporine induced apoptosis The BD ApoAlert Caspase 8 Fluorescent Assay Kits Cat Nos 630218 amp 630219 and BD ApoAlert Caspase 8 Colorimetric Assay Kits Cat Nos 630220 amp 630221 provide assays for caspase 8 protease activity Induction of caspase 8 is one of the earliest events in Fas receptor induced apoptosis Note that staurosporine does not induce caspase 8 Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 3 BD ApoAlert DNA Fragmentation Assay Kit User Manual Introduction continued The BD ApoAlert Annexin V Apoptosis Kits FITC Cat Nos 630109 amp 630110 EGFP Cat No 630113 are based on externalization of phosphatidylserine PS from the inner to the outer leaflet of
19. otocol describes the preparation of formalin fixed paraffin embed ded tissue sections mounted on glass slides For information on fixing and embedding technigues see Ben Sasson et al 1995 Most steps are performed in Coplin jars Section lll You must separate Coplin jars that have been used with DNase from DNase free Coplin jars Note If using fresh frozen tissue sections proceed directly to Step 7 1 2 3 10 11 12 Remove paraffin by immersing the slides in a Coplin jar containing fresh xylene Incubate at room temperature for 5 min Repeat in a second Coplin jar containing fresh xylene Immerse the slides in a Coplin jar containing 100 ethanol and incubate at room temperature for 5 min Rehydrate the slides by seguential 3 min room temperature incuba tions in Coplin jars containing 100 ethanol 95 ethanol 85 ethanol 70 ethanol 50 ethanol Immerse the slides in a Coplin jar containing 0 85 NaCl and incubate at room temperature for 5 min Immerse the slides in a Coplin jar containing PBS and incubate at room temperature for 5 min Fix the slides by immersing them in a Coplin jar containing fresh 4 formaldehyde PBS and incubate at room temperature for 15 min Wash the slides by immersing them in a Coplin jar containing PBS and incubate at room temperature for 5 min Transfer to another Coplin jar containing PBS and incubate at room temperature for 5 min A
20. room temperature Repeat PBS wash Permeabilize the cells by immersing the slides in a Coplin jar containing prechilled 0 2 Triton X 100 PBS Incubate for 5 min on ice Optional Prepare a DNase treated positive control Add 100 ul of DNase Buffer Incubate at room temperature for 5 min Gently tap the slide to remove the liquid Add 100 ul of DNase Buffer containing 0 5 1 ug ml DNase Incubate at room temperature for 10 min Gently tap the slide to remove the liquid Immerse 3 4 times in H O in a Coplin jar reserved for DNase treated samples o oooopo Immerse the slides in a Coplin jar containing fresh PBS for 5 min at room temperature Transfer the slides to a second Coplin jar containing fresh PBS for 5 min at room temperature Proceed to Section VI BD Biosciences Clontech www bdbiosciences com Protocol No PT3137 1 10 Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual V Sample Preparation for Microscopic Detection continued B Cultures of Suspended Cells 1 co 10 11 13 14 15 16 Collect the cells by centrifugation at 300 x g for 10 min at 4 C 2 Aspirate the supernatant carefully 3 Add 500 ul of PBS and gently resuspend the cells 4 5 6 7 Centrifuge the cells at 300 x g for 10 min at 4 C Aspirate the supernatant carefully Resuspend in PBS at a concentration of 2 x 107 cells ml Transfer 5
21. s protein kinases broad spectrum Vincristine Disrupts microtubules Sulfate DNase treated controls Prepare as described in Step V A 8 DNase treatment creates free 3 OH ends that are labeled with fluorescein dUTP Typically gt 70 of treated cells will give a positive fluorescent signal There are two types of negative controls Biological controls Cells or tissues are not subjected to apoptosis inducing treatments TdT minus controls Cells or tissues are not treated with TdT Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 BD ApoAlert DNA Fragmentation Assay Kit User Manual V Sample Preparation for Microscopic Detection A Adherent Cells Most steps are performed in Coplin jars Section lll Keep Coplin jars that have been used with DNase separate from DNase free Coplin jars 1 10 11 Grow cells on Lab Tek Chamber Slides eguivalent commercial slides or on poly L lysine coated slides prepared in your laboratory see Section Ill Induce apoptosis in samples Following induction wash the slides by dipping twice in a Coplin jar containing PBS Use a fresh jar for each wash To fix the cells immerse the slides in a Coplin jar containing fresh 4 formaldehyde PBS at 4 C for 25 min Note You may store fixed cells for up to two weeks at 20 C in 70 ethanol Immerse the slides in a Coplin jar containing fresh PBS for 5 min at
22. the plasma membrane This event occurs shortly after caspase activation Annexin V is a protein with strong affinity for PS The assay is simple and can be used with fluorescence microscopy or flow cytometry The BD ApoAlert Mitochondrial Membrane Sensor Kit Cat No 630106 contains MitoSensor a reagent that detects changes in mito chondrial membrane potential that occur during apoptosis The assay is simple and can be used with fluorescence microscopy or flow cytometry The BD ApoAlertTM Cell Fractionation Kit Cat No 630105 provides a method for isolating a mitochondria enriched fraction from the cytoplasm of apototic and non apototic cells It includes two different antibodies against Cytochrome C The first antibody is used to identify the mitochondria enriched fraction The second antibody is used to determine if Cytochrome C has been released from the mitochondria and is present in the cytosolic fraction an indicator of mitochondrial involvement in apotosis e The BD ApoAlert Caspase 3 Assay Plate Cat Nos 630223 8 630224 is a 96 well plate with the fluorogenic substrate of caspase 3 immobilized in the wells When cell lysate containing active caspase 3 is placed in the wells caspase 3 cleaves the substrate releasing a fluorescent product that can be detected with a standard absorbance microplate reader The BD ApoAlert Caspase Profiling Plate Cat Nos 630225 amp 630226 is a 96 well plate with fluorogen
23. u staining Methods Cell Biol 46 29 39 Cohen J J Duke R C Fadok V A amp Sellins K S 1992 Apoptosis and cell death in immunity Ann Rev Immunol 10 267 293 Ellis R E Yuan J amp Horvitz H R 1991 Mechanisms and function of cell death Ann Rev Cell Biol 7 663 698 Facchinetti A Tessarollo L Mazzocchi M Kingston R Collavo D amp Biasi G 1991 An improved method for the detection of DNA fragmentation J Immunol Methods 136 1251 1256 Gavrieli Y Sherman Y amp Ben Sasson S A 1992 Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation J Cell Biol 119 493 501 Green D R 1998 Apoptotic pathways the roads to ruin Cell 94 695 698 Koester S K Roth P Mikulka W R Schlossman S F Zhang C amp Bolton W E 1997 Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein specific monoclonal antibody APO2 7 Cytometry 29 306 31 2 Nicholson D W 1996 ICE CED3 like proteases as therapeutic targets for the control of inappropriate apoptosis Nature Biotechnol 14 297 301 Nicholson D W amp Thornberry N A 1997 Caspases killer proteases Trends Biochem Sci 22 299 306 Oberhammer F Wilson J W Dive C Morris D Hickman J A Wakeling A E Walker P R amp Sikorska M 1993 Apoptotic death in epithelial cells cleavage of DNA to 300 and or 50
24. will exhibit strong nuclear green fluorescence using a standard fluorescein filter set 520 20 nm All cells stained with PI exhibit strong red cytoplasmic fluorescence when viewed at gt 620 nm Protocol No PT3137 1 www bdbiosciences com BD Biosciences Clontech Version No PR4Y988 15 BD ApoAlert DNA Fragmentation Assay Kit User Manual VII Detection by Flow Cytometry o NOOA 12 17 18 19 20 21 22 23 24 25 Collect 3 5 x 10 cells by centrifugation at 300 x g for 10 min at 4 C Aspirate the supernatant carefully Add 5 ml of PBS and gently resuspend the cells Collect the cells by centrifugation at 300 x g for 10 min at 4 C Aspirate the supernatant carefully Repeat Steps 3 5 Resuspend the cells in 0 5 ml of PBS Fix the cells by adding 5 ml of fresh prechilled 1 formaldehyde PBS Incubate at 4 C for 20 min Collect the cells by centrifugation at 300 x g for 10 min at 4 C 10 11 Carefully aspirate the supernatant Gently resuspend the cells in 5 ml of PBS Collect the cells by centrifugation at 300 x g for 10 min at 4 C 13 14 15 16 Carefully aspirate the supernatant Repeat steps 11 13 Gently resuspend the cells in 0 5 ml of PBS Permeabilize the cells by adding 5 ml of 70 ice cold ethanol Incubate at 20 C for at least 4 hr The cells can be stored at 20 C for up to 1 week Note For a faster procedure permeabilize the
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