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CytoExtr

DNA laddering assay for treated cells


Characteristics of this procedure:
I found the procedure described by Gong et al. to be a convenient and successful method to detect DNA laddering in cells undergoing apoptosis. As the authors describe in their paper (Gong et al., 1994, Anal. Biochem., 218: 314-19), this procedure is simple and uses nontoxic reagents (no phenol, chloroform used). The cells are prefixed in 70% ethanol and, if wished, can be stored in this way for several weeks before analysis. After fixing, the cells are extracted with 0.2 M phosphate-citrate buffer at pH 7.8. Under these conditions, the partially degraded, oligonucleosomal DNA is extracted quite selectively from the cells whereas the higher molecular weight DNA stays associated with the nuclei. The extracts are treated with RNase A and Proteinase K and then loaded to the agarose gel. The authors claim, that DNA laddering can be detected from samples with only 8% apoptotic cells. Alternatively, the cells can be stained with DAPI and analysed by flow cytometry.


PROCEDURE

The protocol is slightly modified from the procedure described in the original paper (only with respect to agarose gel concentration, voltage and running time).

  1. Harvest cells by trypsinization (about 2x106 cells). Spin cells down at 200xg for 5 min.
  2. Resuspend cells in 1 ml Hanks' buffered salt solution (HBSS)
  3. Transfer the cells into 10 ml of ice-cold 70% ethanol; store the cells at -20C for 24 h or longer
  4. Spin down fixed cells at 800 g for 5 min and remove the ethanol thoroughly!
  5. Resuspend the cell pellet in 40 l phosphate-citrate buffer (PCB), consisting of 192 parts of 0.2 M Na2HPO4 and 8 parts of 0.1 M citric acid (pH 7.8). Incubate at RT for at least 30 min.
  6. Spin cells down at 1000 g for 5 min.
  7. Transfer supernatant to new tubes. Optionally, the samples can be concentrated by vacuum in a SpeedVac concentrator.
  8. Add 3 l of 0.25% Nonide NP-40 (in water) and 3 l of RNase (1 mg/ml in water) to the samples and incubate for 30 min at 37C.
  9. Add 3 l of proteinase K (1 mg/ml) to the samples and incubate for another 30 min at 37C.
  10. Add 12 l of loading buffer (0.25% bromophenol blue, 30% glycerol) to the samples and load on a 1.5% agarose gel.
  11. Run the gel at 4 V/cm for about 4 hours and detect DNA by ethidium bromide under UV light.