Paola Bossù
Dompé Research Center, L'Aquila, Italy
EMail: biology@dompe.it

1. Introduction 2. Protocol
  3. Commentary

3.2. Critical parameters


3.3. Troubleshooting


3.4. Anticipated results

3.5. Time considerations 3.6. Key references 1. Wyllie, A.H. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activity. Nature 284: 555.

2. Duke, R.C., and Cohen, J.J. 1986. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolisis. Proc. Natl. Acad. Sci. U.S.A. 80: 6361.

3. Arends, M.J., Morris, R.J., and Wyillie, A.H. 1990. Apoptosis. The role of the endonuclease. Am. J. Pathol. 136: 593.

4. Bortner, C.D., Oldenburg, N.B.E., and Cidlowski, J.A. 1995. The role of DNA fragmentation in apoptosis. Trends Cell Biol. 5: 21.

5. Sellins, K.S., and Cohen, J.J. 1991. Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origin. J. Immunol. 147: 795.

Appendix 1 (A1): Stock solutions

RPMI medium 


RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 mM HEPES buffer, 50 µg/ml gentamicin sulfate. L-glutamine is labile, thus it does not last at 4°C for more than one day. 
TE buffer 10 mM Tris.Cl pH 7.4 (prepare by diluting stock solution), 1 mM EDTA.
Tris.Cl stock solution (1 M) Dissolve 121 g Tris base in 800 ml H2O, adjust to desired pH with concentrated HCl, mix and add H2O to 1 liter. 

CAUTION: Adjust pH of the Tris buffer at the same temperature at which it will be used, as the pH varies with temperature (about 0.028 pH units per 1°C).

Loading buffer 10x 





Prepare concentrated stock solution of loading buffer by adding the following reagents at the indicated final concentrations: 20% Ficoll 400, 0.1 M EDTA (pH 8.0), 1% SDS, 0.25% bromophenol blue, 0.25% xylene cyanol (optional).
TBE buffer stock solution 


Dissolve in 800 ml of H2O 108 g Tris base (89 mM), 55 g boric acid (89 mM), 40 ml 0.5M EDTA, pH 8.0 (2mM); bring to 1 liter with H2O. Use diluted 1:10. 
Ethidium bromide stock solution Dissolve 50 mg of ethidium bromide in 100 ml of H2O. Use diluted 1:1000.
Protect from light.
Agarose gel Dissolve 1% agarose in 1x TBE buffer (in the presence of 0.5 m g/ml ethidium bromide) by heating until melted. 
Prepare just before use.


Appendix 2 (A2): Reagents