Bacterial Membrane Permeabilization Assay

The ability of SP-A (or rfhSP-A), PMB, PMBN, and combinations thereof to permeabilize the outer and cytoplasmic bacterial membranes was studied in live bacteria by quantifying the internalization of the impermeant fluorescent Sytox Green, since its fluorescence increases when binding to bacterial DNA (31 (link)). For the measurement of Sytox Green influx, the probe (1 μM) was added to 1 ml of bacterial suspension (2x10⁷ CFU/ml) in LTM and the sample was incubated for 15 min in darkness at room temperature. Then, the fluorescence of the Sytox Green/bacterial suspension mixture was monitored for 4 hours in a FLUOstar Omega microplate reader (BMG LabTechnologies, Ortenberg, Germany) at excitation and emission wavelengths of 485 and 520 nm, respectively. PBS was used as a negative control, whereas ethanol (70%) was used as a positive control. Background fluorescence was measured in non-labeled bacteria.