This study investigates the interaction between bacterial pathogens and human keratinocytes using a two-chambered infection system. The focus is on the effects of streptolysin S (SLS) on keratinocyte ion transport and subsequent inflammatory responses.
Take a two-chambered infection system separated by a permeable membrane insert. The bottom compartment contains adherent human keratinocytes, or skin cells.
Add bacterial pathogens in the upper chamber, and incubate. The membrane prevents direct contact of the pathogens with the keratinocytes. Pathogens secrete streptolysin S or SLS — a small toxin — that crosses the membrane to reach the cells.
The sodium bicarbonate co-transporter on keratinocytes mediates the influx of bicarbonate and sodium ions, maintaining intracellular pH. SLS binds to the co-transporter and disrupts ion transport, leading to intracellular osmotic stress.
The stress-induced downstream signaling causes degradation of the inhibitor bound to sequestered Nuclear Factor-Kappa B, or NF-κB, leading to its activation. The activated NF-κB translocates to the nucleus, leading to the upregulation of pro-inflammatory cytokine production.
Upon extracellular secretion, the cytokines bind to death receptors on the host cell, initiating necrosis. Post-incubation, harvest the culture supernatant to assess the impact of the bacterial toxin.
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.
繁體中文
