Bacterial Infection Protocols for In Vitro and In Vivo Studies

The MRSA strain USA300/LAC^{53 (link)} was provided by M. Otto (NIH). For infection, USA300/LAC was grown overnight (O/N) at 37 °C in tryptic soy broth (TSB, Sigma) at 250 r.p.m. and was subcultured at a 1:100 dilution for 3.5 h in TSB to mid-log phase. K. pneumoniae strain 43816 serotype 2 was purchased from American Type Culture Collection and was grown O/N at 37 °C in TSB at 250 r.p.m. for infection. S. pneumoniae WU-2 strain from R. Malley (Boston Children's Hospital) was grown at 37 °C under 5% CO₂ without shaking for 18 h in Todd's Hewitt broth (THB, Sigma) with 0.5% yeast extract and was subcultured at a 1:10 dilution for 8 h in fresh THB with 0.5% yeast extract to reach mid-log phase for infection. P. aeruginosa strain PA01V from G. Pier (Brigham and Women's Hospital) was grown O/N at 37 °C in TSB at 250 r.p.m. and was subcultured at a 1:100 dilution for 4 h in TSB for infection. For all strains, cultures were centrifuged at 5,000 r.p.m. for 5 min, and bacterial pellets were washed and resuspended in phosphate-buffered saline (PBS). The OD₆₀₀ was measured to estimate bacterial density, and serial plating was performed on tryptic soy agar (TSA) plates to quantify CFU values. For intravital imaging, a GFP–MRSA S. aureus transgenic bacterium was used, whose construction has been previously reported^{25 (link)}.