1. Introduction
This method is based on the notion that extensively fragmented double-stranded
DNA can be separated from chromosomic DNA upon centrifugal sedimentation.
The protocol includes the lysis of cells and the release of nuclear DNA,
a centrifugation step with the generation of two fractions (corresponding
to intact and fragmented DNA, respectively), precipitation of DNA, hydrolysis
and colorimetrical quantitation upon staining with diphenylamine (DPA),
which binds to deoxyribose.
2.1. Materials
1. Deliver 1.0 ml of cell suspension (not less than 5x105 and no more than 5x106, in order to obtain an OD600 for DNA > 0.04 and < 1.200) in tubes labeled B (bottom).
2. Centrifuge cells at 200xg at 4°C for 10 min.
3. Transfer supernatants carefully in new tubes labeled S (supernatant).
4. Add to the pellet in tubes B 1.0 ml TTE solution and vortex vigorously. This procedure allows the release of fragmented chromatin from nuclei, after cell lysis (due to the presence of Triton X-100 in the TTE solution) and disruption of the nuclear structure (following Mg++ chelation by EDTA in the TTE solution).
5. To separate fragmented DNA from intact chromatin, centrifuge tubes B at 20,000xg for 10 min at 4°C.
6. Transfer supernatants carefully in new tubes labeled T.
7. Add to the small pellet in tubes B 1.0 ml TTE solution.
8. Add 1.0 ml of 25% TCA to tubes T, B and S and vortex vigorously.
9. Allow precipitation to proceed overnight at 4°C.
10. After incubation, recover precipitated DNA by pelleting for 10 min at 20,000xg at 4°C.
11. Discard supernatants by aspiration.
12. Hydrolyze DNA by adding 160 m l of 5% TCA to each pellet and heating 15 min at 90°C in a heating block. Prepare a blank with 160 m l of 5% TCA alone.
13. To each tube add 320 m l of freshly prepared DPA solution, then vortex. Allow color to develop for about 4 h at 37°C or overnight at room temperature.
14. Transfer two 200 m l-aliquots of colored solution (ignoring dark particles) from each tube to a well of a 96-well microtiter plate.
15. Read optical density at 600 nm with a multiwell spectrophotometer reader, setting blank to 0. The excitation wavelength of 600 nm is the optimal one, but wavelengths from 560 to 620 can be used as well.
16. The percentage of fragmented DNA can be calculated using the formula:
S+T+B
where S, T and B are the OD600 of fragmented DNA in the S, T and B fractions, respectively.
17. The fragmented DNA released by cells undergoing apoptosis and lysis during the experiment is recovered in the fraction S and should therefore be taken in consideration in particular circumstances. However, since many substances present in the fraction (serum proteins, componenets of the tisuue culture medium, etc.) could heavily interfere with the OD measurement, is could be more convenient to disregard the S tubes (which most of the times contain only negligible amounts of DNA) and to apply the following formula:
T+B
Apoptosis, or physiological cell death, can be dishinguished from necrosis on the basis of a series of morphological and biochemical parameters. In general, in order to describe a death event as apoptotic, more than one parameters should be evaluated. Among these parameters, DNA fragmentation is very typical of the apoptotic process, with generation within the nucleus of a series of multiplets of a 180 bp subunit, through the action of a Ca++/Mg++-dependent endonuclease with cleaves DNA in the linker region between nucleosome cores.
Measurement of DNA fragmentation with the DPA colorimetric assay is preferentially used to evaluate apoptosis in resting cells or in other cell populations where DNA labeling is impossible or difficult. The method is relatively simple and straightforward.
3.2. Critical parameters
The DPA colorimetric assay requires a relatively high number of cells to obtain reliable results, as at least 1 m g DNA must be measured. One of the most critical drawbacks of the method is the fact that the centrifugal separation technique works only for the extensive typical internucleosomal fragmentation of DNA, but not for rare events of random double-stranded cleavage or single-stranded nicking. Moreover, this method cannot apply to those apoptotic events where no DNA fragmentation occurs.
3.3. Troubleshooting
The main problem of the method is the accurate recovery of fragmented DNA. In fact, depending on the accuracy of the separation step some fragmented DNA can be found in the B fraction. The other problem, as already pointed out, is its lack of sensitivity, as large amounts of DNA (>1 m g) are necessary for each fraction to be detectable. In fact, by using standard calf thymus DNA the OD600 signal is linear between 1.5 m g (OD600 = 0.05) to 50 m g (OD600 = 0.8).
3.4. Anticipated results
The results that can be obtained with this method will give an indication of the precentage of fragmented DNA over the total nuclear DNA of a cell population. No information on apoptosis at the single cell level or at the level of subpopulations can be however obtained with this technique.
3.5. Time considerations
The DPA assay is rather lengthy, compared to other methods to measure DNA fragmentation such as PI labeling and cytofluorimetric analysis. In fact, two days are required to complete the procedure and obtain the final results.
3.6. Key references
2. Cohen, J. J., and Duke, R. C. 1984. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132: 38.
3. Duke R. C., and Cohen, J. J. 1988. The role of nuclear damage in lysis of target cells by cytotoxic T lymphocytes. In: Cytolytic Lymphocytes and Complement: Effectors of the Immune System. E. R. Podak, ed. CRC Press, Boca Raton, FL, pp.35-37.
4. Sellins K. S., and Cohen, J. J. 1987. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J. Immunol. 139: 3199.
5. Wyllie, A. H. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activity. Nature 284: 555.
|
|
|
|
| Complete
RPMI medium
|
RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 mM HEPES buffer, 50 mg/ml gentamicin sulfate. L-glutamine is labile, thus it does not last at 2-8°C for more than one day. | Store at 2-8°C. |
| DPA solution
|
Add 10 ml glacial acetic acid to 150 mg diphenylamine
in a 50 ml polypropylene tube and mix thoroughly by repeated inversion
until complete dissolution. Add 150 m l concentrated
sulfuric acid and mix thoroughly. Add 50 m l
acetaldehyde solution and mix thoroughly.
CAUTION: prepare fresh and use within 60 min. DPA is an irritant: wear appropriate protection. |
|
| Acetaldehyde solution | Add 16 mg acetaldehyde to 10 ml deionized water to have a 16 mg/ml stock solution. | Store at 2-8°C
for < 1 year. |
| 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). |
|
Acetic acid, glacial A-0808
Sigma
Diphenylamine D3409
Sigma
DQ 202 Calf Thymus DNA standard 80-6227-06
Pharmacia Biotech
EDTA disodium salt, dihydrate E-5134
Sigma
FCS A-1111-L
Hyclone
Gentamicin sulfate, solution G-1522
Sigma
L-glutamine, 20 mM, 200 ml 25030-024
GibcoBRL
HEPES sodium salt H-7006
Sigma
RPMI-1640, 500 ml 42402-016
Gibco BRL
Sulfuric Acid S-1526
Sigma
Trichloroacetic acid T-8657
Sigma
Triton X-100 115291A
BioRad
TRIZMA base, T-1503
Sigma
Appendix 3 (A3): Equipment
Multi-block Heater Model 2094
Lab-line Instruments, Inc.
Refrigerated cell centrifuge Model GS-6R Beckman
Refrigerated microcentrifuge Model 5417R
Eppendorf
Vortex Model MT 135
Carlo Erba
Water bath Model 1002
GFL
