Escherichia coli Outer Membrane Vesicle Immunization Protocol and Induction of Bacterial Sepsis    

Materials and Reagents

1. Five-week-old male C57BL/6 or BALB/c mice or a variety of transgenic/knockout mice, body weight 18-20 g
   
2. Phosphate buffered saline (PBS) (Gibco^®, catalog number: 70013-032 )
   
3. Escherichia coli (E. coli) derived OMVs
   
4. E. coli (Isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice)
   
5. Luria-Bertani broth (LB) medium (Merck KGaA, catalog number: 1.10285.0500 )
   

Equipment

1. 1.5 ml microtube
   
2. Ultra-fine-II insulin syringe 1 ml 31 G (0.25 mm x 8 mm) (BD, catalog number: 328820 )
   

Procedure

1. Vaccine preparation
   1. Prepare a suspension containing 10 μg/ml of E.coli OMVs in PBS.
       Notes:
      1. The total amount of the sample depends on the number of mice in the immunized group. For each mouse, 100 μl of sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. If N=5 per group, make a total of 600 μl for each group.
      2. Refer to the protocol " Preparation of Out Membrane Vesicle from Escherichia coli " (Kim et al., 2013b) for preparing OMVs.
   2. Make three aliquots of the above OMVs sample in each 1.5 ml microtube and store at -80 °C until use.
      
      
2. Immunization
   1. At day 0, label the cage (or mouse) to distinguish between immunized and sham.
   2. Bring one of the OMVs sample aliquot to room temperature and mix by vortexing before use.
   3. Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers as in Figure 1 below.
      
      
      Figure 1. Intraperitoneal injection
      
      
3. Bacteria challenge for induction of sepsis
   1. At day 21, prepare E. coli (5 x 10⁹ CFU/ml) in PBS after culturing E. coli in LB broth for 200 rpm at 37 °C until OD₆₀₀=1.0.
      Note: The total amount of the sample depends on the number of mice that will be challenged. For each mouse, 200 μl of the sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. For a total of 10 mice, make 2,100 μl of the sample.
   2. Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers.
   3. Use sterile 1 ml syringe to intraperitoneally inject 200 μl of the E. coli suspension per mouse.
   4. Monitor the survival of mice every 12 h and record.
      Note: The symptoms for sepsis will appear after about 6-8 h of challenge. Sacrifice the mice after 5 days of monitoring. Please refer to the reference article for survival results.
      

Acknowledgments

This protocol was adapted from previously published work (Kim et al., 2013a). The work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).

References

1. Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013a). Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 190(8): 4092-4102.
   
2. Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013b). Preparation of outer membrane vesicle from Escherichia coli. Bio-protocol 3(23): e955.