Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.

<p>NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.</p

Surface marker expression upon stimulation with low LPS concentrations.

<p>Cells were plated and induced as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113840#pone-0113840-g003" target="_blank">Figure 3</a>. After 16 h of induction, cells were harvested and stained for the expression of surface markers using appropriate amounts of fluorescent-labelled antibodies. The median of the fluorescence intensities of 1×10⁴ cells each was recorded by flow cytometry. Fold change values were calculated to compare individual cell types with different compensation settings. Results show mean and SD of at least four independent experiments per cell type. Statistical analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113840#pone-0113840-g003" target="_blank">Figure 3</a>.</p

Measured impurities in protein preparations from different suppliers evaluated by LAL tests.

<p>Measured impurities in protein preparations from different suppliers evaluated by LAL tests.</p

Effects of low LPS concentrations on cytokine production in different human immune cells.

<p><b>A</b> 1×10⁵ THP-1 cells, primary human monocytes, monocyte-derived DCs (moDCs), or CD1c⁺ DCs were either stimulated with different concentrations of LPS or solvent (PBS/BSA) as a control. After 16 h, supernatants were harvested and analysed by ELISA. Results show means and SD of at least four independent experiments per cell type. <b>B</b> Left panel: Cells were plated and stimulated as described above. After 24 h, supernatants were harvested and analysed for IL-1β expression by ELISA. Results show means and SD of at least five independent experiments per cell type. Right panel: 1×10⁵ monocytes were stimulated as described above. After 6 h, cells were harvested and mRNA levels were measured by qRT-PCR. Results show means and SD of three independent experiments. One-way ANOVA with a Dunnett post-test was performed for each cell type individually. * p<0.05, ** p<0.01, *** p<0.001 compared to the solvent control of the respective cell type.</p

Endotoxin sensitivity correlates with CD14 expression.

<p><b>A</b> Expression of TLR4 and CD14 on different human immune cells. Cells were plated and induced as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113840#pone-0113840-g003" target="_blank">Figure 3</a>. After 24 h, cells were stained for the expression of TLR4 and CD14 and analysed by flow cytometry. Results show the percentage of fluorescence-positive cells (% pos cells) compared to the respective isotype controls. Statistical analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113840#pone-0113840-g003" target="_blank">Figure 3</a>. <b>B</b> Analysis of CD1c, CD14 and CD19 expression on primary human CD1c⁺ DCs. 1×10⁵ CD1c⁺ DCs were stained for the expression of CD1c, CD14 or CD19 either directly after isolation (0 h), or after 24 h culture in 24-well plates containing 1 ml of DC-medium. Cells were then analysed by flow cytometry. One representative donor is shown. Light grey, isotype control; dark grey, respective surface marker staining.</p