This protocol describes a real-time assay to assess an imbalance of DNA single-strand break repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye.

Materials :

2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Sigma): 251 mM XTT in DMSO
1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS, Sigma): 0.5 mM 1-methoxy PMS in DMSO (stock solution, -20^oC)
XTT/1-methoxy PMS solution (-20^oC): 3.2 ml XTT solution + 66.6 ml 1-methoxy PMS stock solution
3-aminobenzamide (3-AB, Sigma) 3.3 M in DMSO (stock solution, -20^oC)
3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ, Sigma): 30 mM in DMSO (stock solution, -20^oC)
1x PBS

Protocol description (for one plate) :

1. Count harvested cells (e.g., CHO cells)

2. Prepare cell suspension solution (e.g., 0.35 x 10⁶ cells/6 ml complete medium)

4. Aliquot complete medium without cells (50 ml/well) to 8 wells/plate to prepare blank wells

5. Load cell solution (50 ml/well) to remaining wells of the 96-well plate

6. Incubate cells overnight in regular CO₂ incubator

7. Add complete medium (50 ml/well) to all wells

8. Expose cells to either test chemical or vehicle only (e.g, 1x PBS) for appropriate period

9. Gently aspirate medium from all wells

10. Wash all wells with 1x PBS once

11. Mix 22 ml XTT/1-methoxy PMS solution and 11 ml of complete medium in a 15 ml tube

12. Aliquot complete medium containing XTT/1-methoxy PMS (100 ml/well) to all wells

13. Incubate cells in CO₂ incubator

14. Periodically determine the amount of formazan dye in each well by a 96-well plate reader at 450 nm with 650 nm as the reference filter

15. NAD(P)H depletion (% control) will be calculated as follows: (Absorbance in treated group - blank)/ (Absorbance in control group - blank) x 100

Comments :

1. To test whether NAD(P)H depletion is due to PARP1 activation, add a PARP inhibitor (e.g, 10 mM 3-AB for alkylating agents; 90 mM DPQ for oxidants) 2 hours prior to chemical treatment and keep it in the medium during and after chemical exposure while the cells are analyzed for NAD(P)H levels.

2. Prior to formazan analysis, gently agitate the 96-well plate to mix the contents of each well taking care to avoid generating any bubbles which may interfere with absorbance readings.

3. When handling the 96-well plate, avoid the splashing of medium onto the lid of the plate in order to maintain the appropriate well volume.

4. The number of cells per plate should be optimized to get reasonable absorbance readings.

5. Typical placement of chemical concentrations across a 96-well plate:

References :

Nakamura, J., Asakura, S., Hester, S. D., de Murcia, G., Caldecott, K. W., and Swenberg, J. A. (2003). Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time. Nucleic Acids Res. 31, e104.