Extracting Host Cell Lysate from Infected Host Cells Cultured in a Permeable Membrane System

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 ph