B-lymphocytes have the ability to repair their plasma membranes following injury, such as by bacterial cholesterol-dependent cytolysins. The repair process includes the removal of the pore from the inflicted region of the plasma membrane via lipid raft-mediated internalization. Lipid rafts are critical for B cell receptor (BCR) activation. Cholesterol-dependent pore forming bacterial toxins provide a useful tool for examining the role of lipid rafts in B cell activation and the underlying cellular mechanisms. This method serves as a great alternative of known cholesterol disruption reagents such as filipin, nystatin, and methyl-β-cyclodextrin. Here, we describe a method of damaging primary murine B cell plasma membranes with the Streptococcus pyogenes cytolysin, Streptolysin O (SLO), and monitoring levels of damage, repair and BCR activation.
Keywords: B cell activation; Lipid rafts; Membrane damage; Membrane repair; Streptolysin O (SLO).