Quantification of DNA strand breaks by TUNEL assay in Mycobacterium tuberculosis

Abstract

The primary niche of Mycobacterium tuberculosis (Mtb) is inside phagocytic cells, where the bacteria are exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs). [1], [2] The ROS and RNIs can introduce single-stranded or double-stranded DNA breaks in bacteria. This protocol describes how to quantify the DNA strand breaks in Mtb.

Materials and Reagents

In Situ Cell Death Detection Kit, TMR red (Roche Molecular Biochemicals, Indianapolis, IN, Cat. No.- 12156792910 [3]

Procedure

1. Prepare primary cultures by growing Mtb strains at 37°C in 10 ml 7H9 broth (BD, DIFCO, cat. no.- 271310) supplemented with 0.2% glycerol, 0.1% Tween-80, and ADS (0.5% albumin VWR amresco Life sciences, cat. no. –0332–500 G), 0.2% dextrose, and 0.085% NaCl) with shaking at 180 RPM in a rotary shaker incubator (Lab Therm LT-X; Kuhner, Basel, Switzerland). Please note that assays must be performed in the 7H9 medium supplemented with a catalase-negative supplement (10% albumin-dextrose-sodium chloride (ADS)). Perform the experiment as biological triplicates (n= 3 50 ml tubes).

2. Thaw the cryostocks of Mtb and culture them as described above, until late log phase (OD at 600 nm =1-1.2).

3. From this primary culture, inoculate fresh 7H9 media so that the starting OD=0.1. Allow the Mtb strains to reach log phase (OD at 600 nm =0.6-0.8), which is approximately 3-4 days of incubation.

4. Measure and record OD. Aliquot an equal number of cells (based on OD600) and adjust the volume to 1 ml with complete 7H9 media. 1 ml of 0.8 OD cells were collected in sterile 1.7 ml microcentrifuge tubes.

5. Wash the cells once with sterile 1X phosphate-buffered saline (PBS) by centrifugation (4000 xg for 5 minutes).

6. Resuspend the cell pellet with 2% paraformaldehyde (PFA) in 1X PBS. Always use freshly prepared PFA.

7. Resuspend the cells completely and incubate for at least 60 minutes at room temperature.

8. Centrifuge the tubes at 4000 ×g for 10 minutes and remove the fixative solution completely.

9. Wash cells once with 500 μl sterile PBS.

10. Resuspend the cells in 2% sodium dodecyl sulfate (SDS) in 1X PBS. Incubate the cells for 2 minutes on ice.

11. Centrifuge the tubes at 4000 ×g for 10 minutes and completely remove the SDS completely.

12. Wash cells twice with 500 μl sterile PBS.

13. Resuspend the cells in 100 μl TUNEL reaction mixture (50 μl of Enzyme Solution (Vial 1) to the 450 μl Label Solution in Vial 2). Simultaneously prepare the following control samples:

14. Mix the reaction mix and incubate for 4 hours at +37°C in a humidified atmosphere in the dark. Protect the sample from light.

Note: This step is critical as it will allow the DNA double-strand breaks to be labeled with modified nucleotides (e.g., TMR-dUTP) in an enzymatic reaction. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the template-independent polymerization of deoxyribonucleotides to the 3′-end of single- and double-stranded DNA. This method has also been termed TUNEL (TdT-mediated dUTP-X nick end labeling).

15. Wash samples twice with 500 μl PBS.

16. Resuspend the cells with 200 μl PBS.

17. Analyze the samples directly by flow cytometry. Fluorescence was measured at a fixed emission (585 nm) after excitation with green-yellow laser (561 nm) using a BD FACSAria Flow cytometer (BD Biosciences, San Jose, CA).

18. Acquire a minimum of 10,000 total events per sample.

Data analysis

For the analysis, cells were first gated on singlets (FSC-H vs. FSC-A). Median fluorescence intensity (MFI) of the singlets population in each sample was analyzed and recorded using the BD FACSDiva software.

Validation of protocol

This protocol has been validated in the following research articles:

1. Shee S, Veetil RT, Mohanraj K, Das M, Malhotra N, Bandopadhyay D, Beig H, Birua S, Niphadkar S, Nagarajan SN, Sinha VK, Thakur C, Rajmani RS, Chandra N, Laxman S, Singh M, Samal A, Seshasayee AN, Singh A. Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis. Elife. 2023 Aug 29;12: e80218. doi: 10.7554/eLife.80218. PMID: 37642294 (Figure 3-figure supplement 2b)

2. Shee S, Veetil RT, Mohanraj K, Das M, Malhotra N, Bandopadhyay D, Beig H, Birua S, Niphadkar S, Nagarajan SN, Sinha VK, Thakur C, Rajmani RS, Chandra N, Laxman S, Singh M, Samal A, Seshasayee AN, Singh A. Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis. Elife. 2023 Aug 29;12: e80218. doi: 10.7554/eLife.80218. PMID: 37642294. (Supplementary Figure S3A)

Acknowledgements

This work was supported by Wellcome Trust-Department of Biotechnology (DBT) India Alliance grant IA/S/16/2/502700 (A.S.) and in part by DBT grants BT/PR13522/COE/34/27/2015, BT/PR29098/Med/29/1324/2018, and BT/HRD/NBA/39/07/2018-19 (A.S.), DBT-IISc Partnership Program grant 22-0905-0006-05-987 436, and the Infosys Foundation. A.S. is a senior fellow of Wellcome Trust-DBT India Alliance. S. Shee was supported by a fellowship award from Indian Institute of Science (IISc).

Competing interests

We declare that no competing interests exist.

References

[1]    A. Singh et al., “Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of metabolism-linked redox homeostasis in Mycobacterium tuberculosis,” Mar. 2022, doi: 10.21203/RS.3.RS-1383880/V1.

[2]    S. Shee et al., “Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species,” Antimicrob. Agents Chemother., vol. 66, no. 9, Sep. 2022, doi: 10.1128/aac.00592-22.

[3]    “In Situ Cell Death Detection Kit TMR red Protocol.” Accessed: Aug. 24, 2026. [Online]. Available: https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/in-situ-cell-death-detection-kit-tmr-red-protocol