This study investigates the effects of Streptococcus SLS toxin on mammalian host cells, focusing on the disruption of ion transport and subsequent cellular responses. The methodology includes the use of a permeable membrane system to facilitate the interaction between the bacteria and host cells.
Begin by seeding mammalian host cells in the bottom compartment of a permeable membrane system.
Add Streptococcus — a pathogenic bacteria known to secrete streptolysin S or SLS toxin, to the membrane insert. The SLS toxin diffuses through the membrane pore and reaches the host cell.
The membrane-bound sodium-bicarbonate cotransporters regulate ion transport, maintaining cellular homeostasis. The toxin binds to these cotransporters, disrupting the ion transport and causing osmotic stress in the host cells.
This activates several downstream signaling cascades, including p38 MAPK, a mitogen-activated protein kinase pathway. Activated p38 MAPK phosphorylates its downstream targets, initiating a cellular response against the bacteria.
Next, remove the permeable membrane insert. Aspirate the medium above the host cells. Add lysis buffer to release the cellular contents. Centrifuge to pellet the cell debris.
Collect the supernatant containing soluble lysate components and the pellet containing insoluble lysate components. Store the samples for further analysis.
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.
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