Immediately prior to treatment, use 1X PBS to wash the cells. Then, apply 2 milliliters of fresh growth medium with or without pharmacological treatment to the six-well plates, or 0.5 milliliters of medium to the wells of 24-well plates. Next, under a laminar flow hood, use sterile forceps to carefully place a sterile 0.4-micron permeable membrane insert into each well.
Gentle and sterile handling of the permeable inserts during infection preparation is critical to prevent the membrane from becoming compromised, or contaminated, during this process.
Apply fresh cell culture medium to the upper chamber of each well according to the manufacturer's instructions. Then, apply an appropriate volume of the normalized bacterial cultures to the upper chamber of the permeable membrane insert system and apply bacterial medium to the control wells.
Careful addition of the bacteria to the upper chamber, as well as gentle transport of the infected cells to the incubator, are critical, because it is essential that bacteria do not contaminate the lower chamber during the experimental setup.
Use sterile forceps to carefully remove the permeable membrane insert.
For assessment of host cell lysates, gently aspirate the medium above the monolayer without disturbing the host cells. Then, use PBS to rinse the cells once. Gently aspirate the PBS, and immediately apply a volume of ice-cold lysis buffer to achieve, for example, a protein concentration of 0.5 to 1.5 milligrams per milliliter. Then, incubate the samples on ice for 15 minutes.
Next, use a cell scraper to detach the cells from the plate surface of each well, and transfer the entire contents of each well to a 1.5-milliliter tube. Then, centrifuge the samples at 14,000 RCF and 4 degrees Celsius for 20 minutes.
To assess soluble lysate components, transfer the supernatant to a fresh tube, and store at minus 20 degrees Celsius, or use immediately. To assess nuclear or other insoluble lysate components, reserve the pellet, and store at minus 20 degrees Celsius, or use immediately.
