A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins
Transcrição
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.
To assess for secreted host proteins, use sterile forceps to carefully remove the permeable membrane insert. Then, while not disturbing the monolayer of cells, collect the medium from the lower chamber into 1.5-milliliter tubes.
Centrifuge the samples at 14,000 RCF and 4 degrees Celsius for 10 minutes to pellet cellular debris. Then, remove all but 50 microliters of the supernatant, transfer to a fresh 1.5-milliliter tube, and store at minus 20 degrees Celsius, or use immediately.
