Bacteria were grown overnight in M9 minimal medium containing 0.2% glucose and 0.2% glycerol (M9GG). 50 μl bacterial culture was diluted into 450 μl of minimal medium containing 0.1% casamino acids and 0.2% citrate. M9 minimal medium was supplemented with 1 mM of Spermine NONOate (Enzo Life Sciences) or 100 μM of carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma) when indicated. Then, a sample was taken after 0, 30 or 60 min. The ATP level of each sample was measured using the BacTiter Glo Kit (Promega) according to the manufacturer’s instructions.
Spermine nonoate
Manufactured by Enzo Life Sciences
Sourced in Austria
Spermine NONOate is a chemical compound that acts as a nitric oxide (NO) donor. It is used in research applications to study the biological effects of nitric oxide.
Automatically generated - may contain errors
4 protocols using spermine nonoate
1
Measuring Bacterial ATP Levels
2
Fluorescence Monitoring of Bacterial Metabolism
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Bacteria were grown overnight in M9 minimal medium containing 0.2% glucose and 0.2% glycerol (M9GG). Then, 20 μl of the overnight culture were diluted in 180 μl of minimal medium containing 0.1% casamino acids, 0.2% citrate and 2 μM of Hoechst 33342 in a flat-bottom 96-well plate (Greiner). M9 minimal medium was supplemented with 1 mM of Spermine NONOate (Enzo Life Sciences) or 100 μM of carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma) when indicated. Fluorescence was then monitored every 6 minutes for 30 minutes at 37°C by a plate reader (Tecan LifeScience).
3
Nitric Oxide Donor Preparation Protocol
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All reagents were obtained from Merck (Vienna, Austria) or Sigma (Vienna, Austria), except for proline NONOate (PROLI/NO), diethylamine NONOate (DEA/NO), spermine NONOate (SPER/NO), and S-nitrosoglutathione (GSNO), which were purchased from Enzo Life Sciences (Lausen, Switzerland). PROLI/NO, DEA/NO, and SPER/NO were dissolved in 10 mM NaOH; GSH was dissolved in 1 M NaOH; GSNO was dissolved in 10 mM HCl. Other stock solutions were prepared in ultrapure water (Barnstead, resistance >18 MΩ cm−1).
4
Nitric Oxide Exposure of Mycobacteria
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A 10-ml of static BCG culture was inoculated at an OD600nm of 0.5 in 7H9-OADC or Sauton’s medium, and after 24 h at 37°C, the culture was diluted at an OD600nm of 0.5 and treated at 37°C for 16 h with the NO donor spermine/NONOate (Enzo Life Sciences; 100 μM final). Then, 500 μl of the culture were centrifuged at 2,300 × g and the pellet was resuspended in 1 ml of distilled water for fluorescent dye staining and microscopy. For M. smegmatis, the same conditions were used, except that the culture was treated with spermine/NONOate at an OD600nm of 0.2 instead of 0.5.
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