MycoLight™ Fluorimetric CTC Live Bacteria Quantification Kit

Protocol summary

1. Prepare 10X dye stock solution
2. Prepare bacteria samples
3. Incubate bacteria samples with CTC at 37°C for 30 minutes
4. Analyze sample by flow cytometry with PE-Texas Red channel

Important
Thaw one of each kit component at room temperature before starting the experiment.

1. CTC stock solution (10X):
Add 1 mL of distilled water into the vial of CTC (Component A) to make 10X stock solution.  Note: Stock solution is stable at -20°C for 2 weeks. Protect from light.

1. Prepare bacteria sample with concentration of 10⁶ cells/ml. Grow bacteria into late log phase in appropriate medium. Remove medium by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in appropriate amount of PBS. Note: Measure the optical density of the bacterial culture at wavelength = 600 nm (OD600) to determine the cell number. For E. coli culture, OD600 = 1.0 equals 8 x 10⁸ cells/ml.
   
   
2. Treat cells with test compounds as desired. Remove treatments by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in appropriate amount of Assay Buffer (Component B) so the concentration of bacteria in the treated sample is the same as the live. Note: Determine the concentration of the bacterial culture before starting the treatment. Note: Dead bacteria can serve as negative control.  It is recommended to kill bacteria with 70% ethanol for 30 min followed by 60 min of boiling.
   
   
3. Add 10 µL of the 10X CTC stock solution to 90 µL of the bacterial sample.
   
   
4. Mix well and incubate in the dark for 30 min at 37°C.
   
   
5. Add 400 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.
   
   
6. Monitor fluorescence of bacteria with a flow cytometer through PE-Taxes Red channel (Ex/Em = 488/615 nm). Note: To exclude debris, it is recommended to set the threshold of the flow cytometer as the following: FSC >10,000, SSC>5,000. Note: The efficiency of CTC is highly strain dependent and the staining conditions would be optimized accordingly.