Laurdan

CAPS and AITC (Sigma, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution. Further dilutions were made with ECS or Krebs solution to reach final concentrations of 330 and 100 nM in the case of CAPS and 200 or 100 μM in the case of AITC, respectively. RvD1 an RvD2 were purchased from Cayman Chemical in an ethanolic solution. Laurdan was purchased from Sigma and dissolved in (DMSO) to obtain a 10 mM stock solution. PTX was purchased from Sigma and dissolved in ECS. DMEM, horse serum, fetal bovine albumin, and newborn calf serum were purchased from Gibco (Grand Island, NY, USA). Collagenase, deoxyribonuclease I, poly-D-lysine, and NGF were purchased from Sigma.

Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) is a fluorescent dye that incorporates into lipid bilayers. Its fluorescence is sensitive to changes in the polarity of the environment and is therefore used to report changes of the membrane fluidity. In order to quantify the spectral changes, the Generalized Polarization (GP) value defined by⁶⁷ is calculated for each spectrum. $$GP=\frac{I_{440}-I_{490}}{I_{440}+I_{490}}$$ here, I₄₄₀ and I₄₉₀ are the fluorescence emission intensities at 440 and 490 nm, respectively.
Laurdan (Sigma, Taufkirchen, Germany) was added to the dissolved lipid DOPG in a molar ratio of 1:500. Unilamellar liposomes were prepared as described before. To analyze the effect of nucleotides on the binding of IM30 to DOPG, 1 µM IM30 WT, 0.1 mM liposomes and 2.5 mM GTP (or GDP) were mixed and incubated for 2 h at 25 °C. As the protein was stored in 50% glycerol, the final concentration of glycerol was 15%. For samples without IM30, the corresponding amount of 50% glycerol (20 mM HEPES, pH 7.6) was added.
The fluorescence emission spectra were recorded on a FluoroMax-4 spectrometer (Horiba Scientific, Kyoto, Japan) from 400 to 550 nm with excitation at 350 nm at 25 °C. The slit width was set at 4 nm for excitation and emission of Laurdan.

One millimolar laurdan (Sigma-Aldrich) solution in dimethyl sulfoxide (DMSO) was added to the vesicle suspension in order to have a lipid/probe ratio of 20:1. This mixture was incubated in the dark for 30 min and further diluted in HEPES-buffered saline to a final concentration of 5 μM laurdan and 100 μM phospholipids. Flagella were purified as previously described by mechanical shearing (17 (link)). Increasing quantities of H7 flagella were progressively added to the laurdan/liposome mixture and incubated each time in the dark for 20 min in a circulating water bath at 37°C. Generalized polarization (GP) (23 (link)) was calculated from the emission intensities at 440 nm and 490 nm after excitation at 390 nm, according to the following equation (1): $$\text{GP}=\frac{I_{440}-I_{490}}{I_{440}+I_{490}}$$ using a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). The relative ΔGP was obtained by subtracting the GP value in the absence of flagella from all GP values, for each size of liposome.

Cells were kept in Optimem overnight, mechanically detached by flushing with DPBS (Lonza, Levallois-Perret, France). A suspension of 500 000 cells/ml in DPBS was incubated for 15 min at 37°C with 5 μM Laurdan (Sigma-Aldrich), and fluorescence emissions were determined in the spectrophotometer as above, at an excitation at 365 nm. The Generalized Polarisation (GP) value was determined as: GP = (em₄₄₀−em₄₉₀)/(em₄₄₀+em₄₉₀), em being the emission intensities at the indicated wavelengths [25 (link)].

Horse heart cyt c (type VI, oxidized form), bovine heart cardiolipin (>80% polyunsatured fatty acid content, primary linoleic acid; 98% purity), and laurdan were provided from Sigma Aldrich- Merck KGaA (Darmstadt, Germany) and used without further purification. All reagents were analytical grade.
For mutant cyt c the expression plasmids of horse cyt c (pHCyc) [50 (link)] was subjected to one round of mutagenesis with QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara United States), which introduced Lys72Asn substitution into the horse cyt c gene. Mutant pHCyc plasmids was introduced into E. coli JM 109. Protein expression and purification of the recombinant protein were then conducted as previously described [51 (link)].

The S. epidermidis ATCC 35984 and S. epidermidis 5 were resuspended in PBS to 1.5 × 10⁶ CFU/mL, and 100 μL suspension containing 10 μM 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan, Sigma-Aldrich, USA) was added to a black microplate containing C9 (0 to 128 μg/mL) or benzyl alcohol. After incubation for 1 h, the fluorescence was detected at wavelengths of 350 nm for excitation and 435 nm for emission. The Laurdan generalized polarization (GP) was calculated: GP = (I₄₃₅ − I₄₉₀)/(I₄₃₅ + I₄₉₀), where I₄₃₅ and I₄₉₀ show fluorescence intensity at 435 nm and 490 nm, respectively (36 (link)).