Hepes

Collagen wound models inoculated with PAO1 and incubated for 72 h were sampled as described for the histological analysis using a cork borer and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M HEPES (Sigma) for 1 h. These were washed five times for 5 min in 0.1 M HEPES, stained in 1% OsO₄ in 0.1 M HEPES for 1 h, and then washed for 5 min four times in distilled H₂O. Samples were dehydrated in ethanol and then critical point dried and sputter coated with gold-palladium before examination using an FEI Quanta 250FEG scanning electron microscope (SEM).

HCT116 (ATCC), HT-29 (ECACC) were maintained in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), 1% Penicillin -Streptomycin, 1% non-essential amino acids (Gibco), 1 mM Sodium pyruvate (Gibco) and 10 mM HEPES (Sigma) in a 5% CO₂ incubator. Thp1 XBlue, HEK Null1, TLR2, TLR4, NOD1, and NOD2 bearing NFkB-AP1 reporter (Invivogen) were maintained in the same conditions. Caco-2 (ATCC) were cultured in DMEM supplemented with 10% FBS (Difco), 1% Penicillin-Streptomycin (Gibco), 1% MEM non-essential amino acids (Gibco), 1 mM Sodium pyruvate (Gibco), and 10 mM HEPES (Sigma) in a 10% CO₂. T84 (ATCC) were grown in DMEM-F12 medium supplemented with 10% FBS (Gibco), 1% Penicillin-Streptomycin (Gibco), 1% MEM non-essential amino acids (Gibco), 1 mM Sodium pyruvate (Gibco), and 10 mM HEPES (Sigma) in a 5% CO₂ incubator. Mycoplasma and bacterial contaminations were tested regularly by PCR or using HEK TLR2 cell-line. Cell viability was monitored by MTS measurement using the CellTiter 96 Aqueous One solution (Promega) according to the manufacturer’s recommendations.

For the measurement of cytosolic free calcium levels, 20,000 cells were seeded into 96-well cell culture plates 2 days before measurement. Then, they were washed twice with Hank’s balanced salt solution (HBSS) containing 0.2% BSA and 10 mM HEPES (Sigma-Aldrich). Cells were incubated with 10 μM Fura-2 AM in HBSS containing BSA, HEPES and 0,25% pluronic acid (Sigma-Aldrich) for 60 min at 37 °C, were washed twice and 100 μl HBSS+HEPES+BSA were added to each well. Analyses were performed in a microplate fluorometer with integrated pipetting system (BMG Labtech NOVOstar, Offenburg, Germany) at 340 nm resp. 380 nm for excitation and 510 nm for emission. Cells were allowed to adapt to 37 °C for 10 min before LPA 18:1 (Avanti Polar Lipids Inc., Alabaster, AL, USA), solved in PBS containing 0.2% human serum albumin in indicated concentrations, was added. In cell line experiments, Ki16425 (Sigma-Aldrich) was used as LPA receptor antagonist in concentrations of 0, 2, or 20 μM 1 min prior to the stimulation with LPA. The change in cytosolic free calcium ([Ca²⁺]_i) was calculated by dividing the measured fluorescence intensity at 510 nm with different excitation wavelengths (ratio 340 nm/380 nm). The baseline levels were subtracted. Adenosine triphosphate was used as a positive control, PBS with 0.2% human serum albumin as a negative control.