- Grow an overnight culture in LB broth with shaking
- Wash the bacteria (centrifuge) 2 times with PBS to remove the conditioned-LB. Save and filter sterilize the LB if you want to look at secreted compounds as well
- Adjust the OD to 1 in PBS and aliquot bacteria into 1.5 ml Eppendorf tubes.
- Heat 10 min at 95°C on pre-warmed heat block
- Freeze aliquots of the the OD 1 PBS stocks at -80. Streak some of the heat-killed bacteria on an LB plate and incubate at 37°C for 24-48 hours to ensure there is no growth. Do not use for cell culture experiments until you have verified no growth. Alternatively, inoculate a liquid culture and incubate ≥24 hours
- When you are ready to use the heat killed bacteria, dilute to OD 0.01 (1:100) with media and stimulate your cells with that for inflammation studies. You may have to dilute further depending on the cells you are using.
- This protocol was adapted from Seiler, et al. 2013 J Immunol. 190:1603-1613
Protocol for heat-killing Pseudomonas or MRSA: