Skip to main content
. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: J Innate Immun. 2013 Sep 25;6(2):227–239. doi: 10.1159/000354304

Fig. 5.

Fig. 5

ΔpilA are more susceptible to SP-A-mediated membrane permeabilization. (a) ELF-97® assay. PAO1 and ΔpilA were preincubated with 50 μg/ml hSP-A for 15 minutes before the addition of ELF-97®. Absorbance was measured every 3 minutes at excitation wavelength 355 nm and emission wavelength 535 nm, for a total of 90 minutes. (b) In vitro ThioGlo® assay. PAO1, ΔpilA and ΔpilA comp were preincubated with 50 μg/ml hSP-A for 15 minutes. The bacterial-free supernatants were then mixed with ThioGlo®. Absorbance was measured at excitation wavelength 405 nm and emission wavelength 535 nm. *p < 0.05 when comparing the relative fluorescence unit (RFU) of ΔpilA against PAO1 and ΔpilA comp. (c) Ex vivo ThioGlo® assay. PAO1, ΔpilA and ΔpilA comp were preincubated with 50 μg/ml total protein of BALF from either SP-A+/+ mice or SP-A−/− mice for one hour. The bacterial free supernatants were then mixed with ThioGlo®. Absorbance was measured at excitation wavelength 405 nm and emission wavelength 535 nm. #p < 0.05 when comparing the RFU of ΔpilA against PAO1 and ΔpilA in BALF from SP-A+/+ mice. *p < 0.05 when comparing the RFU of each PA strain in BALF from SP-A+/+ versus SP-A−/− mice. (d) In vitro PA killing by hSP-A. PAO1 and ΔpilA were preincubated with 50 μg/ml hSP-A for one or six hour(s). The bacteria were then stained with a mixture of SYTO 9® and propidium iodide for 15 minutes. The ratio of dead to live bacteria is counted in 10 high power fields. *p < 0.05 when comparing the ratio of dead to live ΔpilA versus PAO1 bacteria in the presence of hSP-A. (e) Ex vivo PA killing by mouse SP-A within BALF. PAO1 and ΔpilA were preincubated with 50 μg/ml total protein of BALF from either SP-A+/+ mice or SP-A−/− mice for one hour. Bacterial viability was determined as in d. #p < 0.05 when comparing the ratio of dead to live bacteria between those treated with BALF from SP-A+/+ versus SP-A−/− mice. *p < 0.05 when comparing the ratio of dead to live PAO1 versus ΔpilA bacteria treated with BALF from SP-A+/+ mice. Experiments were performed independently three times in triplicates (for ELF and ThioGlo assays) or decuplicate (for Live Dead assay). Data from one typical experiment are shown.