This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

FLUOROCHROME LABELLING OF DNA STRAND BREAKS BY TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT-Assay) AND ANALYSIS BY FLOW CYTOMETRY

 

Ildo Nicoletti & Roberta Mannucci
Istituto di Medicina Interna e Scienze Oncologiche
Università di Perugia, 06122 Perugia, Italy
EMail: imiso@unipg.it


Introduction
 
 

Materials  Equipment  
(A1) TdT Staining Solution  
(A2) Rinsing Buffer  
(A3) PI Staining Solution  
Methodology:
  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
 
   Alternative methods:
   
Commentary

Background informations

  Critical parameters   Troubleshooting  

 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.  

  3. 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
  4.  

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