Figure 2.

Preparation of P25/lipopolysaccharide (LPS) and free LPS fractions from a mixture of P25 TiO₂ NP and LPS. (A) The cartoon provides a schematic overview of the experimental approach adopted to obtain LPS-doped P25 nanoparticle (NP). The three fractions (SUP, D. SUP, and P25/LPS suspension) result from the centrifugation of the mixture of LPS (10 ng/ml) + P25 NP (128 µg/ml) in Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS). The P25/LPS suspension is obtained re-suspending 1:250 the P25/LPS pellet in DMEM + 10% FBS so as to obtain the same nominal dose of P25 NP present in the original mixture. For comparison, also the D.SUP has been diluted 1:250. (B) Detection of LPS in extracts from pellets of P25 NP (128 µg/ml) and LPS mixtures. Lanes represent the run of eluates of the following pellets (see Materials and Methods): P25 in DMEM + LPS (10 ng/ml); P25 in DMEM (+10% FBS) + LPS (10 ng/ml); P25 in DMEM + LPS (100 ng/ml); and P25 in DMEM (+10% FBS) + LPS (100 ng/ml). The blot has been performed twice with comparable results. (C) Detection of LPS in western blot. The indicated amounts of LPS, dissolved in Laemmli buffer 4× supplemented with 10% FBS, were treated as described under “Materials and Methods.”