Ap136p

The two cell lines were tested for their basal Cd14 expression and expression after stimulation with LPS (0.1 ng/ml, Sigma-Aldrich, Munich, Germany) for 12 hours. Proteins were isolated from the cells by using NP40 buffer (1 mM PMSF, 1 mM DDT, 1 mM sodium ortho-vanadate, Complete Mini Protease inhibitor cocktail tablet [Roche Applied Science, Mannheim, Germany]) and concentrations were defined by performing Bradford assays. 50 μg protein were used for SDS-PAGE followed by a semi-dry transfer to Optitran BA-S 85 membrane (Sigma-Aldrich, Munich, Germany) using Semi-Dry Blotter Maxi V20-SDB (Carl Roth, Karlsruhe, Germany) and transfer buffer (39 mM glycine, 0.037% SDS, 48 mM Tris, 20% methanol). Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam). After labeling CD14 antibody with goat anti rat IgG [HRP] (AP136P; Millipore, Billerica, USA) and beta actin with F(ab’) 2 rabbit anti mouse IgG [HRP] (STAR21B; AbD serotec, Oxford, UK) as secondary antibodies, luminescence of the different bands was activated by using Roti-Lumin 1 and 2 solution (Carl Roth). Detection of the bands was performed by x-ray film (GE Healthcare, Munich, Germany)

At passage 3, HMSCs were trypsinized, washed, and resuspended in mesenchymal stem cell medium (PromoCell, C-28010) at a concentration of 1 × 10⁵ cells/mL. HMSCs were fixed with paraformaldehyde at 4°C for 15 min and washed with distilled water before permeabilization in 0.5% Triton-X100. Non-specific binding was blocked using blocking solution (PBS containing 1% bovine serum albumin (BSA)) for 1 hour at room temperature. HMSCs were then incubated 2 hours at room temperature with primary antibodies from rabbit against antigrowth associated protein-43 (GAP-43, 1 : 200) (Chemicon, AB5220) and against antiglial fibrillary acidic protein (GFAP, 1 : 500) (Chemicon, AB5804) and from mouse against antineuronal nuclei (NeuN, 1 : 100) (Chemicon, MAB377). After washing, HMSCs were incubated 15 minutes with goat anti-rat IgG (Millipore, AP136P) and goat anti-rabbit IgG (Millipore, 12-348MN) secondary antibodies. After several washes in PBS, HMSCs were incubated with horseradish peroxidase (HRP-) coupled streptavidin for 10 min. DAB (diaminobenzidine) served as chromogen (Figure 2).