Introduction
 
 

The apoptotic process is characterized by activation of nucleases which produce a large number of DNA strand breaks. These breaks can be labelled by attaching to them biotin- or digoxygenin conjugated dUTP in a reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT-assay) or DNA polymerase.
Fluorochrome-conjugated avidin or digoxygenin antibodies are used as second step to label the DNA breaks and more recently, fluorochrome-conjugated deoxy-nucleotides have been made available for a single-step labelling procedure. Detection of breaks requires a pre-fixation with a cross-linking agent, such as paraformaldheyde that unlike ethanol prevents the extraction of fragmented DNA. Cells are then post-fixed in cold ethanol, labelled with nucleotides in a TdT catalysed reaction, resuspended in PBS and counterstained with PI for a simultaneous analysis of cell-cycle and apoptosis.

• Paraformaldehyde 1% in PBS pH 7.4
• Ethanol
• Potassium Cacodylate
• PBS
• TRIS-HCl
• Triton X-100
• BSA
• Cobalt Chloride
• Terminal Transferase (TdT from Sigma or Boheringer)
• FITC-dUTP (or BODIPY-dUTP or CY2-dUTP from Boheringer or Molecular Probes or Amersham)
• RNAse A
• Propidium Iodide (PI) stock solution 50 mg/ml in PBS

• Centrifuge
• Incubator
• Flow Cytometer

• 10 ml of the reaction buffer (1 M Potassium Cacodylate pH 7, 125 mM TRIS-Hcl pH 6.6, 1.25 mg/ml BSA)
• 5 ml of Cobalt Chloride solution (CoCl₂ 25 mM)
• 12.5 units of TdT in its storage buffer (usually 0.5 ml)
• 0.25 nanomoles of FITC-dUTP (or BODIPY-dUTP) in its storage buffer
• Add distilled water to 50 ml

• PBS containing 0.1% Triton X-100 and 5 mg/ml BSA

• Propidium Iodide 5 mg/ml, RNase A 200 mg/ml in PBS

1. Wash cells (1x10⁶) in PBS and centrifuge at 200 g for 5 min
2. Fix cells in 1 ml 1% paraformaldehyde pH 7.4 for 15 min on ice
3. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 5 ml PBS
4. Centrifuge and resuspend cells in 0.5 ml PBS
5. Post-fix the cell suspension in 5 ml ice-cold 70% (vol/vol) ethanol. Leave cells in ethanol for at least one hour (the cells can be stored in ethanol at -20°C for several days)
6. Centrifuge, remove ethanol, resuspend cells in 5 ml PBS and centrifuge
7. Resuspend the cell pellet in 50 ml of TdT staining solution (A1)
8. Incubate cells for 60 min at 37°C
9. Add 1.5 ml of rinsing buffer resuspend throughly and centrifuge
10. Repeat rinsing in 1.5 ml of rinsing buffer (A2) and centrifuge
11. Resuspend cell pellet in 1 ml of PI staining solution (A3)
12. Incubate for at least 30 min at room temperature in the dark and analyse cells by flow cytometry
13. For flow cytometer analysis: construct a dot plot of PI red fluorescence on x-axis vs. green fluorescence of incorporated nucleotides on y-axis
14. Set red (PI) fluorescence acquisition in linear mode
15. Set green (FITC) fluorescence acquisition in logarithmic scale
16. Set photomultiplier parameters (gain and compensation) appropriately (a classic example is provided in figure 1)
17. Run the samples. Collect at least 10⁴ cells/sample

Figure 1. TdT assay in in vitro cultured MCF-7 breast cancer cells and examined by flow cytometry. Nuclei are counterstained with PI to analyse the relationships between cell-cycle position and apoptosis. Contour plot of log-growing cells (left panel) does not show any green fluorescence. Treatment with genistein (100 mM 12h and subsequent 12 h release, middle panel) is followed by the appearance of green-stained (apoptotic) cells. The apoptosis is maximal in the G0-G1 phase of the cell cycle. Continuous (24 h) exposure to the same agent (right panel) produce apoptosis in both G0-G1 and G2/M cell-cycle phases.
 

Alternative methods utilize either digoxigenin-conjugated d-UTP followed by staining with a FITC-conjugated anti-digoxigenin Ab or biotin-conjugated d-UTP followed by staining with FITC-conjugated streptavidin. These methods produce a stronger emission in the green (FITC) channels but they increase the aspecific staining, with a reduced signal/background ratio.

Background informations

• This is the most specific method for evaluation of early phases of apoptosis
• Commercial kits (Boheringer and Amersham) for this assay are available
• Since the DNA content of cells is simultaneously measured, the method provides the unique possibility of analysing the cell-cycle position of apoptotic cells
• An important disadvantage stems from the high cost (particularly when commercially available kits are used)

• A proper fixation to avoid loss of fragmented DNA (the major source of staining!!) is essential
• Maintain the TdT stock solution at –20°C since its activity is critical for nucleotide incorporation into DNA strand breaks

• Necrotic cells and/or cells repairing DNA can present some staining particularly when indirect methods (see alternative methods) are utilized

 Key References
 

1. Gavrieli Y, Y Sherman and SA Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501

 

2. Gorczyca W, J Gong and Z Darzynkiewicz. 1993. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951

 

3. Darzynkiewicz Z, X Li and J Gong. 1994. Assay of cell viability: Discrimination of cells dying by apoptosis. In: Flow cytometry (2^nd Edition). Z Darzynkiewicz, JP Robinson and HA Crissman eds. Academic Press, New York, pp 15-38.