An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

Take a microplate containing mammalian cells on ice.

Add a medium containing calcium to selected wells and a medium without calcium to the others.

Introduce monomers of a bacterial pore-forming toxin. A low temperature prevents monomer aggregation, facilitating binding to cell membrane cholesterol.

Incubate at a physiological temperature, allowing the cholesterol-bound monomers to oligomerize and insert into the membrane, forming a pore.

In wells with extracellular calcium, the pore causes calcium influx.

The influx triggers exosomal shedding of the toxin and membrane resealing.

The intracellular calcium increase also induces lysosomal exocytosis, releasing an enzyme that converts the membrane's sphingomyelin to ceramide.

The ceramide-enriched membrane triggers toxin endocytosis, resealing the membrane.

Membrane resealing is hindered in wells without extracellular calcium.

Add propidium iodide or PI — a fluorescent dye — that cannot enter cells with a repaired membrane but enters damaged cells to bind DNA, emitting fluorescence.

Measure fluorescence at defined intervals to assess the membrane resealing efficiency.