Click-It™ Plus TUNEL Assay for in Situ Apoptosis Detection with Alexa Fluor™ Dyes

USER GUIDE Click-iT™ Plus TUNEL Assay for in situ apoptosis detection with Alexa Fluor™ dyes Catalog Nos. C10617, C10618, C10619 Pub. No. MAN0010877 Rev. B.0 Table 1. Contents and storage Catalog No. Material Concentration Storage* C10617 C10618 C10619 TdT Reaction Buffer 8.0 mL 8.0 mL 8.0 mL 1X Solution (Component A) EdUTP nucleotide mixture 55 µL 55 µL 55 µL 50X Solution (Component B) TdT (terminal deoxynucleotidyl 3 vials (34 µL 3 vials (34 µL 3 vials (34 µL 15 U/μL in transferase) *recombinant* per vial) per vial) per vial) glycerol (Component C) Click-iT™ Plus TUNEL 10X Solution Reaction Buffer, 10X 500 µL 500 µL 500 µL in Tris-buffered (Component D) saline • ≤–20°C • Protect from ™ Click-iT Plus TUNEL light Reaction Buffer Additive 400 mg 400 mg 400 mg NA (Component E) Copper protectant (Component F) 100 µL 100 µL 100 µL NA 1 vial of 1 vial of 1 vial of Alexa Fluor™ picolyl azide dye Alexa Fluor™ 488 Alexa Fluor™ 594 Alexa Fluor™ 647 DMSO (Component G) picolyl azide picolyl azide picolyl azide Solution (25 µL) (25 µL) (25 µL) Proteinase K (Component H) 500 µL 500 µL 500 µL 25X Solution *These storage conditions are appropriate when storing the entire kit upon receipt. For optimal storage conditions for each component, see labels on individual components. NA = Not applicable. Number of assays: Sufficient material is supplied for 50 coverslips based on the protocol below. Approximate fluorescence excitation and emission maxima, in nm: Alexa Fluor™ 488 azide: 495/519 nm; Alexa Fluor™ 594 azide: 590/615 nm; Alexa Fluor™ 647 azide: 650/670 nm; Hoechst 33342: 350/461 nm, when bound to DNA. Note: The insert allows for the convenient storage of all the reagents, including the 1X Click-iT™ Plus TUNEL Supermix, at ≤–20°C. A 15-mL conical tube containing the 1X Click-iT™ Plus TUNEL Supermix will fit into the I slot. For Research Use Only. Not for use in diagnostic procedures. Introduction Understanding the mechanisms of programmed cell death or apoptosis can represent a critical aspect of toxicological profiling and drug discovery. Based on the cellular changes during programmed cell death, apoptosis is often classified into early, middle, and later stages. The later stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and DNA fragmentation. Since the introduction of terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay in 1992,1 the TUNEL assay has become the most widely used in situ test for the study of apoptosis.2 The TUNEL assay is based on the incorporation of modified dUTPs by the enzyme terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA, a hallmark as well as the ultimate determinate of apoptosis. The modifications are fluorophores or haptens, including biotin, which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP) or indirectly with streptavidin or antibodies. System overview The Click-iT™ Plus TUNEL assays utilize EdUTP (a dUTP modified with a small, bio- orthogonal alkyne moiety), which is incorporated at the 3’-OH ends of fragmented DNA by the TdT enzyme. Detection is based on a click reaction,2–5 a copper catalyzed covalent reaction between an Alexa Fluor™ picolyl azide dye and an alkyne (Figure 1). Because of the small size of the alkyne moiety, the EdUTP nucleotide is more readily incorporated by TdT than other modified nucleotides (Figure 2). The mild reaction conditions for the Click-iT™ Plus TUNEL assays have been demonstrated to preserve cell morphology, the binding properties of phalloidin, and the signal from fluorescent proteins such as GFP and RFP (Figures 3 and 4). The Click-iT™ Plus TUNEL assays have been optimized and contain all the components needed to label and detect apoptotic cells from FFPE (formalin-fixed, paraffin embedded) tissue samples or on adherent cells grown on cover slips. The kits include sufficient reagents for labeling fifty (50) 18 × 18 mm coverslips using 50 µL of reaction reagent per test. The kits are flexible and can be configured for 50 independent TUNEL apoptosis tests. Figure 1. Apoptosis detection with the Click-iT™ Plus TUNEL assay. N N H H N E E Wash Wash Wash Nuclear & Fix and Cu(I) optional Image Perm cells (5 min) (5 min) (5 min) staining -N = N+ = N – (Alexa Fluor™ picolyl azide) TdT incorporation of EdUTP into dsDNA Fluorescent detection of strand breaks EdUTP with click chemistry (60 min) (30 min) Click-iT™ Plus TUNEL Assay | 2 Figure 2. Comparison of TdT incorporation of several modified nucleotides. A 48-bp oligonucleotide was incubated with 30 units of TdT and an equimolar mix of the modified nucleotide with three other nucleotides for 4 hours at room temperature. The TdT reaction products were then analyzed by gel electrophoresis; following application to a 20% TBE pre-cast gel and subsequently stained with SYBR™ Gold nucleic acid gel stain. 20 18 16 14 12 10 8 6 4 2 Nucleotide extension (bases per minute) 0 dUTP EdUTP BrdUTP Fluorescein- BODIPY™ Biotin- dUTP FL-dUTP dUTP Figure 3. Formalin-fixed, paraffin embedded (FFPE) mouse intestine was treated with DNAse to fragment the DNA. After treatment, the Click-iT™ Plus TUNEL assay with the Alexa Fluor™ 594 dye (red in the figure below) was utilized to detect the fragment DNA. After the TUNEL reaction, actin was stained with Alexa Fluor™ 647 phalloidin (pink). The cells were counterstained with Hoechst 33342 (blue). The GFP expressing epithelial cells are clearly visible (green). Click-iT™ Plus TUNEL Assay | 3 Figure 4. HeLa cells were transduced with CellLight™ Mitochondria-RFP, BacMam 2.0 and treated with DNase to induce TUNEL positive DNA strand breaks. After the treatment, the Click-iT™ Plus TUNEL assay with the Alexa Fluor™ 647 dye was utilized to detect the fragment DNA (purple). After the TUNEL reaction, actin filaments were stained with ActinGreen™ 488 ReadyProbes™ Reagent (green). The RFP has localized to the mitochondria, resulting in the red fluorescent signal. Before you begin Materials required but not provided • 1X Phosphate buffered saline (PBS) (Cat. No. 14190-144 or 14190-250) • 4% paraformaldehyde in PBS (fixative) • 0.25% Triton™ X-100 in PBS (permeabilization reagent) • 5 mg/mL Bovine serum albumin and 0.1% Triton™ X-100 in PBS, pH 7.4 • 1X solution of Hoechst 33342 • 3% Bovine serum albumin in PBS (3% BSA in PBS), pH 7.4 • Molecular biology grade water (DNase/RNase free) • 22 × 22-mm or 18 × 18-mm coverslips (for standard microscopy) • DNase I (Cat. No. 18068-015) Cautions TdT reaction buffer (Component A) contains potassium cacodylate and cobalt chloride, and it is harmful if swallowed. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. If swallowed, seek medical advice immediately. Wear appropriate laboratory protective clothing, gloves, and eye/face protection when handling this reagent. Protocols This user guide includes protocols to perform the Click-iT™ Plus TUNEL imaging assay on FFPE tissue samples or adherent cells grown on coverslips Click-iT™ Plus TUNEL Assay | 4 Prepare the stock solutions 1.1 Allow vials to warm to room temperature before opening. 1.2 Prepare a working solution of 1X Click-iT™ Plus TUNEL Reaction buffer (Component D): Transfer all of the solution (500 µL) in the Component D vial to 4.5 mL of deionized water. Rinse the Component D vial with some of the diluted Click-iT™ Plus TUNEL Reaction buffer to ensure the transfer of all of the 10X concentrate. To make smaller amounts of 1X Click-iT™ Plus TUNEL Reaction buffer, dilute volumes from the Component D bottle 1:10 with deionized water. After use, store any remaining 1X solution at 2–8˚C. When stored as directed, this 1X solution is stable for up to 6 months. 1.3 Prepare a working solution of 1X Click-iT™ Plus TUNEL Supermix according to Table 2. Table 2. Click-iT™ Plus TUNEL Supermix. Picolyl azide Picolyl azide Picolyl azide Supermix components Alexa Fluor™ 488 Alexa Fluor™ 594 Alexa Fluor™ 647 1X Click-iT™ Plus TUNEL 2630 µL 2625 µL 2625 µL Reaction Buffer (from Step 1.2) Copper Protectant 67 μL 67 μL 67 μL (Component F) Alexa Fluor™ picolyl azide 3.7 μL 8.3 μL 8.3 μL (Component G) TOTAL Volume 2.7 mL 2.7 mL 2.7 mL After use, store any remaining 1X solution at ≤–20°C. When stored as directed, this 1X solution is stable for up to 6 months. 1.4 To prepare a 100X stock solution of the Click-iT™ Plus TUNEL Reaction Buffer Additive (Component E): Transfer 2 mL of deionized water to the contents of the vial (400 mg), then mix until fully dissolved. After use, aliquot any remaining stock solution and store at ≤–20°C. 1.5 For tissue samples only: Prepare a 1X Proteinase K solution by diluting Component H 1:25 in PBS. After use, aliquot any remaining stock solution and store at ≤–20°C. Experimental protocol for tissue sections The following protocol was developed using FFPE tissue sections of mouse intestine, kidney, liver, heart, and colon. The tissue type and treatment may influence the number of apoptotic cells detected. Deparaffinization tissue sections 2.1 Deparaffinize tissue sections in Coplin jars at room temperature according to Table 3. Table 3. Tissue deparaffinization procedure. 50%:50% 100% 100% 95% 85% 75% 50% 0.85% 1X Xylenes Xylenes Xylenes:EtOH EtOH EtOH EtOH EtOH EtOH EtOH NaCl PBS 5 min 5 min 3 min 5 min 3 min 3 min 3 min 3 min 3 min 5 min 5 min Click-iT™ Plus TUNEL Assay | 5 Fix and permeabilize tissue sections 3.1 Immerse the slides in fixative (4% paraformaldehyde) for 15 minutes at 37°C.

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