3. Commentary
Apoptosis is an innate mechanism of eukariotic cell suicide which plays a major role in many physiological and pathological processes. Therefore, the definition of cellular regulatory mechanisms and biochemical processes involved in apoptosis is an important challenge from both theoretical and applied points of view.
During apoptosis a series of reorganisation occur in the cell: chromatin condensation, loss of cell volume and membrane blebbing are some of the most evident morphological changes of apoptotic cells. Although the molecular mechanisms leading to such changes are not completely known, many of them seem to proceed in parallel with biochemical events. This is the case, for example, of chromatin condensation and nuclear envelop breakdown. In fact, in parallel with them occurs DNA fragmentation, a biochemical hallmark of apoptosis in the majority of cells. Responsible for DNA cleavage is believed to be an endogenous Ca++- and Mg++-dependent endonuclease able to break double strand DNA at internucleosomal sites. Therefore, apoptotic DNA cleavage results in characteristic fragments of oligonucleosomal size (180-200 bp). Such phenomenum, described for the first time by Wyllie (1980), can be visualized by an agarose gel electrophoresis analysis. The present protocol provides a method for qualitative determination of DNA fragmentation.
3.2. Critical parameters
The most critical point of DNA electrophoretical analysis is its inability of quantitative measurement of apoptosis. In fact, due to problems linked to the insolubility of large DNA and thus to its final agarose gel analysis, the method is strictly qualitative.
Moreover, this method is not recommended when different behaviour in DNA fragmentation following apoptotic stimuli is described. In fact, in some cell types where random double-stranded or rare single-stranded DNA fragmentation occur, it cannot be detected by agarose gel electrophoresis assay.
Sometimes, in particular with cells obtained from ex vivo cultures (e.g. thymocytes and lymphocytes), an high background of spontaneous DNA fragmentation could be observed.
3.3. Troubleshooting
Solubilization of chromosome-length DNA collected in tubes B is generally difficult. Increase of TE volumes and extension of incubation time may be needed for the redissolution of DNA following precipitation. The use of limited number of cells (less than 5x106) will be helpful to limit this problem. However, since the method is exclusively qualitative, the analysis of fragmented DNA present in tubes T is the main interest of the assay.
3.4. Anticipated results
The assay of DNA agarose gel electrophoresis provides good results for the definition of cell apoptosis. A typical ladder pattern of DNA fragmentation should be observed in most apoptotic cells.
3.5. Time considerations
Preparation of DNA for agarose gel electrophoresis analysis depends on the DNA size of samples: generally from 2 to 7 days are required. Additional 4-8 hr are needed for performing electrophoresis.
3.6. Key references
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
| Complete 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. | |
| Agarose gel | Dissolve 1% agarose in 1x TBE buffer (in the presence of 0.5 m g/ml ethidium bromide) by heating until melted. |
Appendix 2 (A2): Reagents
, 500 ml
42402-016
Gibco BRL
Gentamicin sulfate, solution G-1522
Sigma
L-glutamin 20 mM, 200 ml 25030-024
Gibco BRL
FCS A-1111-L
Hyclone
EDTA disodium salt, dihydrate E-5134
Sigma
TRIZMA base (Tris) T-1503
Sigma
Triton X-100 115291A
BioRad
Sodium Chloride S-9888
Sigma
Isopropyl alcohol 412421
Carlo Erba
Ethanol 1170
Riedel-deHaen
Lauryl sulphate, sodium salt (SDS) L-4390
Sigma
Ficoll 400 F-4375
Sigma
Bromophenol blue B-5525
Sigma
Xylene Cyanol X-4126
Sigma
Boric acid B-0394
Sigma
Ethidium bromide E-7637
Sigma
DNA molecular weight markers IX 1449460
Boehringer Mannheim
Agarose standard 18054
Eurobio
Polaroid film type 667 F-4638
Sigma
Appendix 3 (A3): Equipment
Multi-block Heater Model 2094
Lab-line Instruments, Inc.
Refrigerated cell centrifuge Model GS-56R
Beckman
Refrigerated microcentrifuge Model 5417R
Eppendorf
Vortex Model MT 135
Carlo Erba
Water bath Model 1002
GFL
Gel electrophoresis apparatus Model Horizon 58
Gibco BRL
Power supply Model 1000/500
BioRad
UV Transilluminator Model T2202
Sigma
Direct screen instant camera Model DS34
Polaroid
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