TUNEL Apoptosis Detection Kit (Green Fluorescence)

Transcription

1 TUNEL Apoptosis Detection Kit (Green Fluorescence) Item NO. KTA2010 Product Name TUNEL Apoptosis Detection Kit (Green Fluorescence) ATTENTION For laboratory research use only. Not for clinical or diagnostic use Version

2 TABLE OF CONTENTS INTRODUCTION Background & Principle... 1 Storage/Stability... 1 Assay restrictions... 1 PRODUCT INFORMATION Materials supplied and Storage conditions... 2 Other supplies required, Not Supplied... 2 Precautions... 2 Technical hints... 2 ASSAY PROTOCOL Recommended procedures... 3 Version

3 INTRODUCTION Background & Principle One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the 3 -hydroxyl termini in the double-strand DNA breaks generated during apoptosis. The TUNEL assay relies on the presence of nicks in the DNA which can be identified by TdT, an enzyme that catalyzes the addition of dutps that are labeled with fluorescein. This kit provides all the essential components with an optimized assay protocol, suitable for fluorescence microplate reader, fluorescence microscope, or flow cytometer. Its signal can be easily detected at the popular FITC channel (Ex/Em = 490 nm/520 nm). Storage/Stability Stable for at least 6 months at -20 o C from date of shipment. Once opened, refer to list of materials supplied for storage conditions of individual components. Gel pack with blue ice. Assay Restrictions Assay kit is intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Cautions: The reaction buffer contains cacodylate and cobalt chloride, highly toxic chemicals. After contact with skin, wash immediately with plenty of water. In case of accident or if you feel unwell, seek medical advice immediately. Do not drink, eat or smoke when using. 1

4 PRODUCT INFORMATION Materials supplied and Storage conditions Kit components Quantity 20T 50T 100T Storage conditions TdT Enzyme 20 µl 50 µl 100 µl -20 C Equilibration Buffer (5 x) 0.5 ml 1 ml 2 ml -20 C Label Mix Green 100 µl 250 µl 500 µl -20 C protect from light Other supplies required, Not Supplied Microcentrifuge Pipettes and pipette tips Phosphate-buffered saline (PBS) Fluorescence microscope Technical hints To avoid cross-contamination, change pipette tips between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Make sure all necessary equipment is switched on and set at the appropriate temperature. 2

5 ASSAY PROTOCOL Recommended procedures A. Sample Preparation 1. For adherent cells (Analysis by Fluorescence microscope) a. Grown in a 96-well microplate culture for at least 24h. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. b. Remove the medium and fix the cells with 4% paraformaldehyde in PBS for 30 min at room temperature. c. Remove the fixation solution and wash with PBS three times (5 min each time). d. Add permeabilization reagent (0.2% Triton X-100 in PBS, not supplied) after the fixation, and incubate the plate for 30 minutes at room temperature. e. Wash the cells with PBS 2-3 times. Proceed with Step B 1. Optional: You may also prepare a positive control for TUNEL reaction using by digesting cells with 10 U/mL DNAase I for 10 min at room temperature before proceeding to TUNEL reaction (Step B 1). 2. For non-adherent cells (Analysis by Flow Cytometry) a. Culture cells to an optimal density (about 1 to 2 x 10 6 cells/ml). Induce apoptosis by desired methods. Concurrently incubate a control culture without induction. b. Collect 1-5 x 10 6 cells by centrifugation at 300g. Wash with 0.5 ml of PBS twice. c. Add 1 ml of 4% paraformaldehyde (in PBS, ph 7.4) and incubate on ice for 30 min. d. Centrifuge the cells at 300g. Remove the supernatant and resuspend in 1 ml PBS. Repeat this wash twice. e. Resuspend cells in 0.3% Triton-X 100 for 5 min at room temperature to permeabilize (Alternatively, resuspend the cells in 100 g/ml Proteinase K for 5 min to permeabilize). f. Centrifuge the cells at 300g. Remove the supernatant and resuspend in 1 ml PBS. Repeat this wash twice and Proceed with Step B For Paraffin-Embedded Tissue (Analysis by Fluorescence microscope) a. Deparaffinize tissue by immersing twice in xylene for min. b. Rehydrate tissue by the following washes (in the order given): two washes for 5 min 3

6 each in 100% ethanol, then one wash for 3 min each successively in 95, 70, and 50% ethanol. c. Wash the sample in PBS twice for 5 min each. d. Drain excess PBS from tissue and incubate for 15 min in 20 µg/ml Proteinase K (in PBS) solution. Note: The time of protease digestion will have to be optimized for specific tissue types and thicknesses. Overdigestion by protease will result in loss of cellular structure and possible release of tissue section from slide. Underdigestion will result in poor TdT labeling. Make protease solutions fresh just before use. e. Terminate the protease treatment by washing cells three times for 5 min each in PBS with gentle agitation. Proceed with Step B For Frozen tissue sections (Analysis by Fluorescence microscope) a. After sections have dried on the slide, fix with 4% paraformaldehyde in PBS for 30min at room temperature. b. Wash by immersing in PBS twice for 5 min each. c. Drain excess PBS from tissue and incubate for 15 min in 20 µg/ml Proteinase K (in PBS) solution. d. Terminate the protease treatment by washing cells three times for 5 min each in PBS with gentle agitation. Proceed with Step B 1. B. Tunel assay 1. Analysis by Fluorescence microscope a. Prepare TdT labeling reaction buffer just before use based on the number of samples to be assayed: Reaction Components Volume Per Well TdT Enzyme 1 µl Equilibration Buffer (5 x) 10 µl Label Mix Green 5 µl ddh 2O 34 µl Total volume 50 µl b. Add 50 µl of the reaction mixture (from Step A 1, 3 and 4) to each sample and incubate at 37 o C for 60 minutes. c. Wash samples three times for 5 min each in PBS. d. Counterstain sample by incubating in 1 µg/ml propidium idodide or 0.05 µg/ml DAPI in PBS for 10 min. 4

7 Note: Concentration of counterstain may have to be adjusted depending on the tissue being stained. Overstaining by propidium iodide or DAPI may result in difficulty in observing the fluorescein label. e. Wash sample three times for 5 min each in PBS. f. Add an aqueous mounting medium or an antifade solution, mount a coverslip and analyze using fluorescent microscopy with a fluorescein filter. 2. Analysis by Flow Cytometry a. Resuspend cells in 100 µl of 1 x Equilibration Buffer. Incubate at room temperature for 10 min. b. Centrifuge cells at 300g. Remove the supernatant and resuspend in 50 µl of TdT labeling reaction buffer. Incubate at 37 o C for 60 min. Periodically mix cells gently. c. Centrifuge cells at 300g. Remove the supernatant and resuspend in 1 ml PBS. Repeat wash twice. d. Resuspend in 1 ml 1 µg/ml propidium idodide or 0.05 µg/ml DAPI in PBS. Incubate 10 min. e. Analyze cells by flow cytometry. 5