TiterTACS 96-well Apoptosis Detection Kit Catalog Number: TA600 Colorimetric Kit 96 tests This package insert must be read in its entirety before using this product. FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES
Contents TABLE OF CONTENTS Page PRINCIPLE OF THE ASSAY 2 REAGENTS PROVIDED........................................................... 2 MATERIALS REQUIRED BUT NOT PROVIDED 3 Reagents.................................................................. 3 Equipment 3 Disposables................................................................ 3 PROCEDURES 4 Reagent Preparation......................................................... 4 Assay Protocol 6 Sample Preparation and Fixation............................................... 6 Labeling Procedure 8 Controls.................................................................. 9 DATA INTERPRETATION 9 TROUBLESHOOTING GUIDE...................................................... 11 APPENDICES 12 Reagent and Buffer Composition.............................................. 12 Fixation Methods 13 Storage.................................................................. 13 Assembling a Humidity Chamber 13 WARNINGS.................................................................... 14 Hazardous Ingredients 14 Handling Precautions.......................................................14 Emergency Exposure Procedures 14 MANUFACTURED FOR AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place NE (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 656-4400 United States of America E-MAIL: info@rndsystems.com R&D Systems Europe, Ltd 19 Barton Lane TELEPHONE: +44 (0)1235 529449 Abingdon Science Park FAX: +44 (0)1235 533420 Abingdon, OX14 3NB E-MAIL: info@rndsystems.co.uk United Kingdom R&D Systems GmbH FREEPHONE: (0) 800 909 4455 Borsigstrasse 7 TELEPHONE: +49 (0)6122 90980 65205 Wiesbaden-Nordenstadt FAX: +49 (0)6122 909819 Germany E-MAIL: infogmbh@rndsystems.co.uk R&D Systems Europe 77 boulevard Vauban FREEPHONE: +0800 90 72 49 59041 LILLE CEDEX FAX: +0800 77 16 68 France E-MAIL: info@rndsystems.co.uk
PRINCIPLE OF THE ASSAY For many cell types in culture, identification of apoptosis can be achieved using a combination of morphological criteria, extraction and analysis of DNA by agarose gel electrophoresis and in situ detection of DNA fragmentation in immobilized cells. Additional approaches include measuring the activation of ICE-like proteases, detection of annexin binding at the cell surface and cleavage of poly-adp ribose polymerase. However, in many cases, the treatment and analysis of a large number of samples is inconvenient. TiterTACS kits are designed specifically for the in situ detection of apoptosis in suspension and monolayer cell cultures. TiterTACS kits provide quantification of apoptosis in cultured cells without direct counting of labeled cells. The TiterTACS 96-well plate assay allows detection of apoptosis in situ using colorimetric detection. TiterTACS kits provide all the reagents for the detection of DNA fragmentation in cells grown as a monolayer or in suspension. TACS Nuclease allows positive controls to be generated for each experimental system: a brief treatment of cells with the TACS-Nuclease prior to labeling generates DNA breaks in every cell, providing an appropriate positive control specific for the system under study. REAGENTS PROVIDED TA600 TiterTACS Component # Component Quantity Storage Conditions 4876-60-01 Cytonin 6 ml 2-8 C 4821-96-01 Proteinase K Solution 100 L -20 C** 4817-60-02 10X TdT Labeling Buffer* 20 ml 2-8 C 4817-60-03 10X TdT Stop Buffer 20 ml 2-8 C 4821-96-04 TdT dntp Mix 35 L -20 C** 4821-96-05 TdT Enzyme 35 L -20 C** 4821-96-14 50X Mn 2+ 100 L -20 C** 4800-30-06 Streptavidin-HRP 30 L 2-8 C 4800-30-12 Blue-Strep Diluent 7.5 ml 2-8 C 4822-96-08 TACS-Sapphire 10 ml 2-8 C 4800-30-15 TACS-Nuclease 15 L 2-8 C 4800-30-16 TACS-Nuclease Buffer 1.5 ml 2-8 C *Contains 0.01% thimerosal. **Store in a manual defrost freezer. Prior to opening the vial, centrifuge for 15-20 seconds at 3000 rpm. 2
MATERIALS REQUIRED BUT NOT PROVIDED Reagents 10X phosphate-buffered saline (PBS), ph 7.4 (Ca 2+ and Mg 2+ free) 37% formaldehyde 100% ethanol or denatured alcohol Methanol Sucrose Deionized water, DNase-free Tween 20 30% hydrogen peroxide (H 2 O 2 ) 50% phosphoric acid or 2 N HCl Equipment 37 C incubator Microplate reader (450 nm and 630 nm filters) Centrifuge (with microplate adaptors) Pipettes and pipette tips Humidity chamber Ice bucket Disposables 96 well microplate Microcentrifuge tubes 10 ml serological pipettes 15 ml tubes Gloves 3
PROCEDURES Reagent Preparation Reagents marked with an asterisk (*) should be prepared immediately before use. The volumes given for each reagent are based on processing samples in a 96-well microplate. If conical well plates are used, the volume of solution may be decreased to 30 µl per well. 1. 1X PBS Refer to the appendix for preparation of 10X PBS. Approximately 250 ml of 1X PBS is used to process 96 samples and to make 1X PBS with 0.1% Tween 20. Dilute 10X PBS to 1X using DNase-free water. Store 1X PBS at room temperature. 2. 1X PBS with 0.1% Tween 20 Add 0.1% Tween 20 to 1X PBS. Approximately 100 ml is required to wash the plate in Step 14. 3. 3.7% Buffered Formaldehyde* 20 ml of fixative is used to process 96 samples. To prepare, add: Sucrose 1X PBS 10 g 40 ml 37% Formaldehyde 5 ml Adjust volume to 50 ml with 1X PBS and store at room temperature. Wear gloves and exercise caution when handling formaldehyde solutions. 4. Proteinase K Solution* 50 µl of Proteinase K Solution is used per sample. Thaw Proteinase K at room temperature, then place on ice until use. Note: Prior to opening the Proteinase K Solution, centrifuge for 15-20 seconds at 3000 rpm. Prepare immediately before use: DNase-free water Proteinase K 50 L 1 L 5. Cytonin If required, 50 µl of Cytonin is used per sample. Cytonin is ready to use. Store at 2-8 C. Discard if solution is cloudy. 6. 2.5% H 2 O 2 Solution* 5 ml of H 2 O 2 solution is necessary per 96 samples. To prepare 6 ml of solution, mix: 30% H 2 O 2 0.5 ml Methanol 5.5 ml 7. 1X TdT Labeling Buffer 25 ml of 1X Labeling Buffer is used to process 96 samples. Dilute the 10X TdT Labeling Buffer to 1X using DNase-free water. Store at room temperature until use. Remove an aliquot of 50 µl per sample for preparing the Labeling Reaction Mix (step 7) and place on ice. 4
8. Labeling Reaction Mix* Thaw the TdT dntp Mix at room temperature, then place on ice. To maintain optimal enzyme activity, remove the TdT Enzyme from the freezer only long enough to pipet the required volume. Alternatively, place the TdT Enzyme in a -20 C freezer block. Prepare the Labeling Reaction Mix just before use and keep the prepared Reaction Mix on ice. Prepare one sample without the enzyme (refer to Controls section). Prepare 50 µl per sample. Note: Prior to opening the TdT Enzyme, centrifuge for 15-20 seconds at 3000 rpm. 96 samples n samples TdT dntp Mix 35 L n x 0.35 L 1X TdT Labeling Buffer (step 6) 5000 L n x 50 L 50X Mn 2+ 100 L n x 1 L TdT Enzyme 35 L n x 0.35 L 9. 1X TdT Stop Buffer 20 ml of 1X TdT Stop Buffer is used to process 96 samples. Dilute the 2 ml of 10X TdT Stop Buffer to 1X with 18 ml DNase-free water. Store at room temperature until use. 10. Streptavidin-HRP Solution* 50 µl of Streptavidin-HRP Solution (1:1250) is used per sample. Note: Prior to opening the Streptavidin-HRP, centrifuge for 15-20 seconds at 3000 rpm. For 96 samples n samples Blue-Strep Diluent 5 ml n x 50 L Streptavidin-HRP 4 L n x 0.04 L 11. TACS-Sapphire TACS-Sapphire is ready to use. Warm to room temperature and add 100 µl of solution per well. Protect from light. 12. Nuclease Solution* 50 µl of Nuclease Solution is required for each Nuclease-treated control sample. Prepare Nuclease Solution just before use and place on ice. Note: Prior to opening the TACS-Nuclease, centrifuge for 15-20 seconds at 3000 rpm. 1 sample TACS-Nuclease Buffer TACS-Nuclease 50 L 1 L 13. Stop Solution 50% Phosphoric Acid or 2 N HCl - Add 100 µl per well to stop the colorimetric reaction. *Prepare reagents immediately before use. 5
Assay Protocol It is important to read through the instructions for use before preparing cell samples for labeling. There are key steps included that are very important for successful labeling. This section includes instructions for Sample Preparation, in situ Labeling and Plate Reading. Prior to labeling, the samples must be fixed and washed in PBS. The Labeling Procedure begins with samples in PBS regardless of the fixation and immobilization method. Sample Preparation and Fixation Preparation of Cells in Suspension Cells grown in suspension or prepared from dissociated tissues, can be fixed in batch solution and then transferred to 96-well plates for duplicate or triplicate analysis or may be grown and fixed directly in the 96-well plates. Batch Method: 1. Harvest the cell suspension by centrifugation at 500 x g for 5 minutes at room temperature. Prepare enough cells for your assay. Typically, 2.0 x 10 4-1.0 x 10 5 cells/well will generate sufficient signal. 2. Wash cells in 1X PBS and centrifuge. 3. Discard PBS and resuspend cell pellet at 1.0 x 10 6 cells/ml in 3.7% Buffered Formaldehyde Solution (see Reagent Preparation section). Let stand for 7 minutes at room temperature. Note: Do not leave longer than 10 minutes. 4. Centrifuge cells at 500 x g for 5 minutes at room temperature and discard fixative. 5. Wash cell pellet once with 1X PBS, centrifuge and discard PBS. 6. Post-fix sample in 100% methanol for 20 minutes at room temperature. 7. Wash cell pellet twice with 1X PBS. Centrifuge between washes. 8. Resuspend cells at 1.0 x 10 6 cells/ml in 1X PBS. 9. Distribute cells at 2.0 x 10 4-1.0 x 10 5 cells/well. The exact cell number should be determined empirically. 10. Proceed to step 1 of the Labeling Protocol (page 9). 6
In Well Method: 1. Distribute or grow 2.0 x 10 4-1.0 x 10 5 cells/well in a 96-well microplate. 2. Centrifuge plate at 1000 x g for 3 minutes at room temperature and discard media. 3. Wash cell pellets once with 200 L of 1X PBS at room temperature, centrifuge and discard PBS. 4. Fill wells with 3.7% Buffered Formaldehyde Solution. Let stand for 7 minutes at room temperature. Note: Do not leave longer than 10 minutes. 5. Centrifuge plates at 1000 x g for 3 minutes at room temperature and discard fixative. 6. Wash cell pellets with 200 L of 1X PBS, centrifuge and discard PBS. 7. Post-fix samples in 100% methanol for 20 minutes at room temperature. 8. Wash cells twice with 200 L of 1X PBS. Centrifuge between washes. 9. Proceed to step 2 of the Labeling Procedure (page 9). Preparation of Cells in Monolayer Method: 1. Centrifuge plate at 1000 x g for 3 minutes at room temperature and discard the media. 2. Wash cells twice with 200 L of 1X PBS at room temperature. Centrifuge between washes. 3. Fill wells with 3.7% Buffered Formaldehyde Solution. Let stand for 7 minutes at room temperature. Note: Do not leave longer than 10 minutes. 4. Centrifuge as in step 1 and discard fixative. 5. Wash cells twice with 200 L of 1X PBS at room temperature. Centrifuge between washes. 6. Post-fix samples in 100% methanol for 20 minutes at room temperature. 7. Wash cells twice with 200 L of 1X PBS. Centrifuge between washes. 8. Proceed to step 2 of the Labeling Procedure (page 9). 9. For storage options, refer to the Appendix (page 14). 7
Labeling Procedure Prepare solutions required as directed in the Reagent Preparation section. Note: After centrifugation steps, supernate can be removed by inverting plate or by pipetting. Step Instructions Notes 1. Wash cells with 200 ml of 1X PBS per well, centrifuge, and discard PBS. 2. Add 50 L of Proteinase K Solution per well and incubate for 15 minutes at room temperature. Samples can also be treated with 50 L/well of Cytonin as an alternative to Proteinase K. Cytonin is recommended for cells in monolayer. 3. Centrifuge plate at 1000 x g for 3 minutes at room temperature and discard buffer. 4. Wash once with 200 L/well of DNase-free water. Repeat step 3. 5. Generate a positive control using TACS-Nuclease at this point. Other samples may be covered with PBS during preparation of the positive nuclease-treated control. After DNase-free water wash, add 50 L of Nuclease Solution to each control well and incubate for 10-60 minutes at 37 C. 6. Wash samples for 2 minutes in 200 L of 1X PBS. Repeat step 3. 7. Quench endogenous peroxidase. Add 50 L/well 2.5% H 2 O 2 solution and incubate for 5 minutes at room temperature. Do not exceed 5 minutes. 8. Wash once with 200 L/well of DNase-free water. Repeat step 3. 9. Add 150 L/well of 1X TdT Labeling Buffer. Incubate for 5 minutes at room temperature. 10. Repeat step 3. 11. Add 50 L/well of Labeling Reaction Mix and incubate at 37 C for 1 hour. 12. Add 150 L/well of 1X TdT Stop Buffer for 5 minutes to stop labeling reaction. 13. Wash samples twice with 200 L of 1X PBS for 2 minutes per wash. Use a humidity chamber during incubation (see the Appendix) or use a microplate cover. Repeat step 3. Centrifuge plate between each wash (refer to step 3). 14. Add 50 L/well of Streptavidin-HRP solution and incubate at room temperature for 10 minutes. 15. Wash samples four times with 200 L/well of PBS with 0.1% Tween 20. 16. Add 100 L/well of TACS-Sapphire at room temperature. Centrifuge plate between each wash (refer to step 3). If working with suspension cells, ensure that cells are resuspended. 17. Incubate at room temperature for 30 minutes in the dark. Follow kinetics of reaction at 630 nm to determine the linear range of the reaction. 18. Stop reaction with 100 L of 50% Phosphoric acid or 2 N HCl per well. Measure absorbance at 450 nm. Read plate within 30 minutes of acid addition. 8
Controls Note: The controls that should be included when performing the protocol for the first time are listed below. Controls should be run in duplicate or triplicate. TACS Nuclease-treated Control Treat 2 or 3 samples with TACS-Nuclease to generate DNA breaks in every cell. The TACS Nuclease-treated controls will confirm that the permeabilization and labeling reaction has succeeded. The information can help optimize the conditions for the labeling procedure. The colorimetric readings obtained with this control will be higher than the experimental values and will provide a maximum value. Unlabeled Experimental Control Sample The TdT Enzyme should be omitted from the Labeling Reaction Mix for 2 samples. These controls will indicate the level of background labeling associated with non-specific binding of the Streptavidin-HRP. These controls should have low or negligible absorbance. Experimental Negative Control Sample An appropriate experimental control should be included in each experiment and will depend upon the system under study. Typically, the Experimental Negative Control will be an untreated sample or normal cells. Many normal or untreated cells will have a small number of apoptotic cells resulting in a low level of labeling. DATA INTERPRETATION Duplicate or triplicate samples will allow statistical validation of results. The suggested controls are important in data interpretation and allow optimization of in situ detection of apoptosis without expending valuable test samples. Refer to the Troubleshooting Guide for information if the controls do not provide the expected results. Figure 1 and Figure 2 show typical results obtained with this TiterTACS kit: Figure 1: Quantitation of Apoptosis in Staurosporine-treated ML-1 cells using TiterTACS. Apoptosis was detected in fixed ML-1 cells after treatment with 1 µm staurosporine for 24 hours. Cells were harvested, fixed and labeled according to the TiterTACS protocol prior to colorimetric analysis. Cells were incubated with TACS-Sapphire substrate and the colorimetric reaction was stopped with 2 N HCl after 30 minutes. The percentage of apoptotic cells in the culture was estimated by the enumeration using the TACS TdT-DAB Apoptosis Detection Kit (R&D Systems, Catalog # TA4625). The cell culture was diluted with a non-apoptotic cell culture to obtain the different concentrations of apoptotic cells for the assay. 9
Figure 2: Quantitation of Apoptosis in Staurosporine-Treated ML-1 Cells Using TiterTACS. Data obtained after stopping the reaction with 50% phosphoric acid, 30 minutes after addition of substrate. Control wells were untreated (without apoptosis inducer), unlabeled (without TdT enzyme) and nuclease-treated cells. All the control wells contained 1.0 x 10 5 cells. Note: Experimental values displayed in the graph show the linearity of the reaction. Experimental results may vary depending on the type of cells, length and condition of storage and cell treatment. Morphological observation of the cells is recommended prior to the assay. 10
TROUBLESHOOTING GUIDE Note: Rule out major problems by checking the labeling in the control samples first. Problem Possible Cause Solution No labeling in TACS-Nuclease treated sample. No labeling in experimental sample. Excessive background in negative control. Poor permeabilization and/or excessive fixation with cross-linking fixative preventing enzyme access. No DNA left in sample due to hydrolysis (poor storage of samples). Excessive (removed all DNA) or inadequate Nuclease treatment. TdT Enzyme is inactive. The enzyme is the most labile component in the kit. No apoptosis (or necrosis) occurring in sample. Residual unlinked Streptavidin-HRP. Optimize Proteinase K treatment or optimize time in Cytonin, reduce time in fixative to 5 minutes. Read the section Preparation and Storage of Samples prior to labeling. Optimize time for Nuclease treatment (5 minutes up to 2 hours). TdT Enzyme must be stored at -20 C (in a manual defrost freezer). Place in a -20 C freezer block or remove aliquot from tube directly in the freezer. If all controls gave the expected results and were processed at the same time as the experimental sample, there may be no DNA fragmentation in cells within the sample. Always examine the morphology of cells. Wash cells at least 4 times with 1X PBS, 0.1% Tween 20. Non-specific binding of Streptavidin-HRP. Incubate Streptavidin-HRP with a blocking reagent such as 5% (w/v) non-fat dry milk or fetal bovine serum in 1X PBS, 0.1% Tween 20. Poor duplicate or triplicate values. Insufficient centrifugation or poor removal of buffer. Inaccurate pipetting. Loss of cells during washes. Centrifuge after every wash. Use care when removing buffer. Use a conical 96-well plate to perform the assay on suspension cells. Transfer to a flat bottom plate after incubation with TACS-Sapphire. 11
APPENDICES Reagent and Buffer Composition 10X PBS, ph 7.4 25 mm sodium dihydrogen phosphate (NaH 2 PO 4 ) 75 mm disodium hydrogen phosphate (Na 2 HPO 4 ) 1.45 M sodium chloride (NaCl) Deionized, sterile water Cytonin Note: Water used should be DNase-free. Distilled autoclaved water can be used. Proprietary permeabilization reagent TACS-Nuclease Proprietary endonuclease TACS-Nuclease Buffer 50 mm Tris-HCl, ph 8.0 1 mm MgCl 2 100 µg/ml BSA 10X TdT Labeling Buffer 1 M TACS Safe-TdT Buffer (proprietary) 0.5 mg/ml BSA 0.6 mm 2-mercaptoethanesulfonic acid (MESNA) 10X TdT Stop Buffer 0.1 M EDTA, ph 8.0 TdT dntp Mix 0.25 mm Biotinylated dntp 7.5 mm dntps TACS-Sapphire Non-toxic, non-organic peroxidase substrate 12
Fixation Methods There are several fixation methods commonly used that are appropriate for the protocol described in the instructions. Formaldehyde is the recommended fixative based on laboratory testing. However, other fixatives that maintain DNA integrity may be used. These include alcohol fixatives such as ethanol, methanol or acetone and other cross-linking agents including paraformaldehyde and glutaraldehyde. Regardless of the fixative used, it is important not to fix cells for extended periods of times. Fixatives other than formaldehyde should be empirically tested to ensure cells can be labeled post-fixation. Storage Long term storage: After fixation and post-fixation steps, cells can be stored in 80% ethanol at 2-8 C or -20 C for several weeks or months. If cells are stored in a microplate, an adhesive or plastic cover is recommended to prevent contamination or evaporation. For labeling after storage, wash the cells with 80% ethanol, then wash three times with 1X PBS and proceed with the Labeling Protocol. Note: When cells are fixed using alcohol (e.g. ethanol), signal intensity in positive cells may diminish with time due to loss of small DNA fragments. Short term storage: The cells can be stored in Cytonin at 2-8 C for up to one month. An adhesive plate cover is recommended to prevent contamination and evaporation. Proceed with step 2 (Labeling Procedure, p. 9) after permeabilization. Assembling a Humidity Chamber To prevent evaporation, it is recommended that incubations at 37 C be carried out in a humidity chamber. A humidity chamber can be made using a plastic box with a tight-fitting lid and two glass rods or other support. Place a paper towel on the bottom of the box and wet thoroughly with water. Lay the glass rods parallel to each other and less than one 96-well plate length apart on the wet tissue. Position the plate on glass rods and place the plastic box, with lid, in a 37 C incubator. Ensure that the plate is horizontal. 13
WARNINGS Hazardous Ingredients The acute and chronic effects of overexposure to reagents of this kit are unknown. One reagent contains minute amounts of thimerosal, which as a concentrated solution may be fatal if swallowed, inhaled, or absorbed through the skin. Thimerosal contains mercury; abide by local regulations for handling and disposal. Handling Precautions Safe laboratory procedures should be followed when handling all kit reagents. It is recommended that protective laboratory clothing and equipment (gloves, laboratory coat, safety glasses) be worn when handling kit reagents. Emergency Exposure Procedures In case of exposure to reagent solutions, we recommend following these emergency first-aid procedures: Skin or eye contact Wash with water for at least 15 minutes. Remove any contaminated clothing. Inhalation Remove individual to fresh air. If breathing is difficult, give oxygen and call a physician. Ingestion Rinse mouth with copious amounts of water and call a physician. TACS, TACS-Nuclease, TACS-Sapphire, and Cytonin are trademarks of Trevigen, Inc. Tween is a registered trademark of ICI Americas Inc. 14
NOTES 2008 R&D Systems, Inc. 03.99 750391.9 4/08 15