Maximum binding of differently loaded CD1d dimers.

<p>(A+B) Highest relative staining reached with each CD1d dimer loaded with indicated glycolipid as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.g002" target="_blank">Fig 2</a>. Presented (A) as absolute or (B) as relative to the highest value reached for each CD1d. Human CD1d: Binding maxima are for all glycolipids are reached with Tyloxapol as surfactant. Mouse CD1d: Binding maxima with αGC and PBS57 are reached with Tween 20, forDB01-1 with Triton X-100 and for PBS44 with Tyloxapol. Rat CD1d: Binding maxima for αGC, DB01-1 and PBS44 are reached with Triton X-100, and for PBS57 with Tyloxapol. Cotton Rat: All maxima reached using Tyloxapol. Statistical analysis can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s003" target="_blank">S2 Table</a>. Hierarchy of surfactant efficacy between experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>. Data were computed from results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.g002" target="_blank">Fig 2</a>.</p

<i>In vitro</i> loading analysis of rat CD1d tetramers.

<p>PE conjugated CD1d tetramers were incubated with 40x molar excess of indicated glycolipid in presence of 0.05% indicated surfactant or without surfactant o.n. at 37°C. After <i>in vitro</i> loading, the indicated amount of CD1d tetramers was used for staining of 10⁵ iNKT TCR transduced cells. Binding presented as relative staining, i.e. ratio between geometric means of CD1d tetramer and CD3 antibody staining performed simultaneously. In total, staining was performed three times independently using oligomers derived from two independent loadings. Hierarchy of surfactant efficacy between experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>.</p

<i>In vitro</i> loading analysis of CD1d dimers.

<p>CD1d dimers were incubated with 40x molar excess of indicated glycolipid in presence of 0.05% of indicated surfactant or without surfactant o.n. at 37°C. After <i>in vitro</i> loading, the indicated amount of CD1d dimers was used for staining of 10⁵ iNKT TCR transduced cells, dimer binding was revealed by staining with PE labeled donkey F(ab′)₂ fragment anti-mouse IgG (H+L). Binding presented as relative staining, i.e. ratio between geometric means of CD1d dimer and CD3 antibody staining was performed simultaneously. In total, staining was performed three times independently using oligomers derived from two independent loadings. Hierarchy of surfactant efficacy between experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>. Note that scales differ in order to facilitate visualization different hierarchies of binding caused by the different surfactants.</p

Experimental setup.

<p>(A) α1 and α2 domain amino acids of CD1d molecules used in this study. Alignment was performed using BioEdit. Human: GenBank BC027926.1, Mouse: GenBank AK002582.1, Rat: GenBank AB029486.1, Cotton Rat: GenBank KM_267558. (B) <i>In vitro</i> loading analysis of CD1d dimers presented as schematic overview. (C) Scheme explaining the emergence of different saturating curves depending on loading efficacy of dimers. Upper panel: Inefficient loading leads to low proportion of functionally bivalent CD1d dimers, Lower panel: Efficient loading leads to high proportion of functional bivalent CD1d dimers. Drawing represents several step flow cytometry analysis of incubation/wash/detection. (D) Structural features of all glycolipids used in this study.</p