Supplemental Digital Content 1
Methods
LAURDAN Generalized polarization function
For the LAURDAN GP measurements the fluorescence signal were collected on the
microscope in two different channels simultaneously. The experimental setup is described
in detail in[1]. The fluorescence signal was split between two PMTs (Hamamatsu H7422P40) by a dichromatic mirror splitting at 475 nm. The PMTs had bandpass filters mounted
438  12nm and 494  10 nm respectively.
LAURDAN generalized polarization (GP) measurements [2] were used to detect and
evaluate the organization of the extracellular lipids within the cholesteatoma and the
normal skin stratum corneum, as described by [3]. LAURDAN is an amphiphilic probe that
preferentially partitions into lipid membranes. The fluorescence emission properties are
sensitive to the water content and dynamics that occurs in the probe’s environment.
Briefly, the energy of the LAURDAN excited singlet state progressively decreases when the
extent of water dipolar relaxation process is augmented, red shifting the probe’s emission
spectrum, i.e. decreasing the GP function to lower values (see equation 1). The extent of
water dipolar relaxation is normally related to the lipid packing of the studied membrane
being low when the membrane packing is tight (e.g. membranes in the gel phase) [1].
Likewise, LAURDAN GPs were calculated using the formula:
GP 
I B  G  I R 
I B  G  I R 
(1)
where IB and IR correspond to the intensities at the blue and red edges of the emission
spectrum (440 and 490 nm) using a given excitation wavelength [1, 4]. In lipid bilayers
high LAURDAN GP values (0.5 – 0.6) correspond to laterally ordered phases (e.g solidordered or gel) whereas low LAURDAN GP values (below 0.1) correspond to liquiddisordered -like phases [1, 2]. The correction factor G was calculated by acquiring GP
images of a known LAURDAN reference solution in the microscope at the same
instrumental conditions used in the tissue sections (for further details see [1]). LAURDAN
(2µM) in DMSO was used, with the predetermined GP=0.011. The reference’s GP was
measured as described [1].
Lipid extraction and lipid analyses
In these experiments, the lipids were extracted from10 individual cholesteatoma samples.
The control samples obtained from normal human skin stratum corneum (see materials
section) were collected from at least 6 individuals.
Samples of human skin stratum corneum from breast reduction surgery were isolated after
overnight floating on 1% trypsin solution (cell culture grade). Cells from the viable
epidermis were removed from stratum corneum sheets by repeated rigorous shaking. The
cholesteatomas were rinsed thoroughly in 0.9% NaCl (by weight) immediately after
surgery to avoid contamination with blood. Each sample preparation was checked under
the microscope to ensure lack of viable epidermal cells. All samples, i.e. cholesteatoma and
human skin stratum corneum were rinsed in 0.88% KCl (by weight) prior to
homogenization and homogenized for 3 x 1 minutes using an UltraThurrax® at max speed.
Samples were dried and weighed. Lipids were extracted by a modified Bligh and Dyer [5]
method: Dry tissue was suspended in a total volume of 1.9mL chloroform:methanol:0.88%
KClaq 2:1:0.8 (by volume). Following rigorous mixing and sonication, the ratio of solvents
was changed to chloroform:methanol:0.88% KClaq 1:1:0.9 (by volume), and samples were
again rigorously mixed. Following centrifugation the solution divides into an upper
aqueous phase and a lower organic phase. The lower (organic) phase was transferred to a
new tube and the upper (aqueous) phase was re-extracted twice with fresh solvent from
the lower phase of the chloroform:methanol:0.88% KClaq 1:1:0.9 (by volume) mixture. The
combined lower phases were washed once with fresh upper phase from the
chloroform:methanol:0.88% KClaq 1:1:0.9 (by volume) mixture and dried under a stream of
nitrogen. Lipids were resuspended in the lower phase of the chloroform:methanol:0.88%
KClaq 1:1:0.9 (by volume) mixture and filtered through a solvent resistant 0.45µm filter
prior to analysis.
High performance thin layer chromatography (HPTLC) analyses:
Lipid extracts were diluted according to dry tissue weight and extracts from individual
samples corresponding to 150µg starting material (tissue dry weight) was loaded onto the
silica plates under a stream of nitrogen. Plates were developed with chloroform-acetonemethanol 76:4:20 (first up to 10 mm, followed by up to 25 mm), then chloroform-acetonemethanol 80:10:10 (up to 75 mm) as the second, and chloroform-diethyl ether-ethyl
acetate-methanol 72:6:20:2 (up to 90 mm) as the third developing system. Cholesterol
containing lipids were visualized by staining plates with 10%FeCl3 in 5% CH3COOH and 5%
H2SO4 (pink/purple for cholesterol) followed by baking at 100°C for 2-7 minutes.
Remaining lipids on the plates were stained with 10% CuSO4 in 8% H2PO3 followed by
baking at 180°C for 10-15 minutes. Standards included for identification of co-migrating
bands were: Cholesterol and cholesterol sulphate (SigmaAldrich), Nu-Chek 18-4A standard
(Nu-Chek Prep, Inc.; which contains equal amounts (by weight) of cholesterol, oleic acid,
methyl olein, triolein, cholesteryl ester), and a free fatty acid mix (FFA) contain equal
amounts (by weight) of C19-C26 fatty acids acquired separately from Larodan Sweden.
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