Recombinant MD-2 binds to live bacteria. (A) The indicated live bacteria were left untreated (shaded profiles) or were opsonized with MD-2^6xHis (solid line) and subjected to cytofluorimetry using an Alexa 647–labeled α-6xHis mAb. The Nm lpxA⁻ strain is LPS⁻. (B) Live Yp cells were incubated with MD-2^6xHis, and surface-bound MD-2 was detected by cytofluorimetry using either TLR4-Fc or an α–MD-2 mAb followed by a FITC-labeled α-mouse antiserum (solid-line profiles). The shaded profiles represent the binding of the secondary reagent without MD-2 coating. Fluorescence intensity is plotted in a log scale (x-axis, 10-10⁵). (C) Live Yp were adsorbed to high-protein-binding plates in either fixed amounts (2 × 10⁷/well; left panel) or in 2-fold dilutions (right panel). MD-2^6xHis was then applied to the wells in 2-fold dilutions (left panel) or in a fixed amount (20 ng/mL; right panel). MD-2 bound to adsorbed bacteria was detected by ELISA using an α–MD-2 mAb. Results are shown as the average plus SD of triplicate absorbance readings at 450 nm. (D) Yp (Kim5) was left untreated (left panel) or was incubated with MD-2^6xHis as in panel A (right panel). MD-2^6xHis was stained with an α-6xHis mAb and imaged by SEM. Shown are the back scatter images of 2 random fields acquired at 8000×. White bar equals 1 μm.