4.6. Bacterial Membrane Permeabilization Assays

The ability of SP-A and SP-B^N, alone and mixed, to permeabilize the outer and cytoplasmic membranes of K. pneumoniae K2 was evaluated by quantifying (i) the fluorescence intensity of DPH and (ii) the uptake of Sytox Green. Exponential-phase bacteria (1 × 10⁷ CFU/mL) were treated with SP-A (100 µg/mL) and/or SP-B^N (10 µg/mL) at 37 °C for 30 min in PBS buffer. The suspension was incubated with 20 μM of DPH dissolved in N,N-dimethylformamide for 1 h at 37 °C in darkness. DPH fluorescence intensity was measured using an SLM-Aminco AB-2 spectrofluorimeter equipped with a thermostated cuvette holder (Thermo Spectronic, Waltham, MA, USA). Quartz cuvettes of 5 mm path length were used. The excitation and emission wavelengths were 350 and 450 nm, respectively [30]. Non-labeled bacteria were used as the background. All experiments were conducted in triplicate.

For the measurement of Sytox Green influx, the probe (1.25 μM) was added to 1 mL of K. pneumoniae suspension (2 × 10⁷ CFU/mL) in PBS and the sample was incubated for 15 min in darkness at room temperature. Then, 100 µL of the Sytox Green/bacterial suspension mixture was added to each well of a 96-well black microplate containing SP-A, SP-B^N, and mixtures thereof (final concentrations of SP-A and SP-B^N: 100 and 10 µg/mL, respectively, with 1 × 10⁷ CFU/mL of bacteria) and fluorescence was monitored for 30 min at 37 °C in a FLUOstar Galaxy microplate reader (BMG Lab Technologies, Ortenberg, Germany) with excitation and emission wavelengths of 485 and 520 nm, respectively [28]. PBS was used as a negative control, whereas ethanol (70%) was used as a positive control [28]. Non-labeled bacteria were used as the background. All experiments were conducted in triplicate.