MycoLight™ Cyclic-di-GMP Red Sensor

1. Grow bacteria in a desired growth medium.
2. Add MycoLight™ Cyclic-di-GMP Red Sensor working solution.
3. Incubate cells for 10 to 30 minutes at RT.
4. Monitor fluorescence in Cy5 or violet filter set 

Note: Before first use, thaw MycoLight™ Cyclic-di-GMP Red Sensor at room temperature and centrifuge briefly to collect the dried pellet. 

Prepare 10 to 20 mM stock solution in DMSO. e.g. Add 210 μL of DMSO into MycoLight™ Cyclic-di-GMP Red Sensor vial to make 10 mM stock solution. 

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Prepare 10 to 20 µM MycoLight™ Cyclic-di-GMP Red Sensor working solution. For example, add 2 μL of 10 mM MycoLight™ Cyclic-di-GMP Red Sensor stock solution into 998 μL of 1X PBS buffer (for live bacteria staining) or 10 mM Tris-HCl, pH 7.5 containing 60 mM KCl (for solution based assay) to make 20 µM MycoLight™ Cyclic-di-GMP Red Sensor working solution. 

Note: Protect the working solution from light by covering it with foil or placing it in the dark.

Note: For best results, this solution should be used within a few hours of its preparation.

The following protocols only provides a guideline and should be modified according to your specific needs.

1. Grow bacteria in growth medium as desired.
2. Treat cells as desired to induce Cyclic-di-GMP.
3. Centrifuge bacterial cells at 10,000 RPM for 10 mins to remove the growth medium and wash bacterial cells with 1X PBS.
4. Add 100 µL of MycoLight™ Cyclic-di-GMP Red Sensor working solution to cells.
5. Incubate cells for 10 to 30 minutes at RT, protected from light.
6. Monitor fluorescence with fluorescence microscope using Violet or Cy5 filter set (best Ex/Em = 490/650 nm) .

Note: The concentration and incubation time of MycoLight™ Cyclic-di-GMP Red Sensor used varies with different bacterial strains, one will need test with different concentrations to get the optimal dose.

Table 1. Layout of Cyclic-di-GMP standards and test samples in a solid black 96-well microplate.

Table 2. Reagent composition for each well.

1. Prepare cyclic-di-GMP standards (cGMP, not provided), blank controls (BL), and test samples (TS) according to the layout provided in tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
2. Add 50 µL of MycoLight™ Cyclic-di-GMP Red Sensor working solution to each well of cyclic-di-GMP standards, blank control, and test samples to make the assay volume of 100 µL/well. For a 384-well plate, add 25 µL into each well instead, for a total volume of 50 µL/well.
3. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/650 nm (cut off at 590 nm).