Direct observation to chemokine receptor 5 on T-lymphocyte cell surface using fluorescent metal nanoprobes

Abstract

Chemokine receptor 5 (CCR5) is a cell surface protein required for HIV-1 infection. It is important to detect the amount and observe the spatial distribution of the CCR5 receptors on the cell surfaces. In this report, we describes the metal nanoparticles which were specially designed as molecular fluorescent probes for imaging of CCR5 receptors on the T-lymphocytic PM1 cell surfaces. These CCR5 monoclonal antibodies (mAbs) metal complexes were prepared by labeling mAbs with Alexa Fluor 680 followed by covalent binding the labeled mAbs on the 20 nm silver nanoparticles. Compared with the labeled mAbs without metal, the mAb-metal complexes were found to display enhanced emission intensity and shortened lifetime due to interactions between fluorophores and metal. The mAb-metal complexes were incubated with the PM1 cell lines. The confocal fluorescent intensity and lifetime cell images were recorded on single cells. It was observed that the mAb-metal complexes could be clearly distinguished from the cellular autofluorescence. By analyzing a pool of cell images, we observed that most CCR5 receptors appeared as clusters on the cell surfaces. The fluorophore-metal complexes developed in this report are generally useful for detection of cell surface receptors and provide a new class of probe to study the interaction between the CCR5 receptors with viral gp120 during HIV infections.

Figure 1

Alexa Fluor 680 labeled anti-CCR5 mAb or mAb-metal complex were immuno-interacted with target CCR5 receptor on T-lymphocytic PM1 cell.

Figure 2

Representative time traces of single Alexa Fluor 680, fluorescent anti-CCR5 mAb, and mAb-metal complex upon excitation at 635 nm. The insets represent the respective typical single molecule or nanoparticle fluorescence images recorded in either emission intensity or lifetime. The scales of diagrams were 5 × 5 μm and the resolutions were 100 × 100 pixel with an integration of 0.6 ms/pixel.

Figure 3

Histograms of emission intensity and lifetime collected from the fluorescence images of labeled mAbs in the absence of metal and mAb-metal complexes.

Figure 4

Upper panel: representative single PM1 cell images that were collected from the cells under the incubation with different concentrations of mAb-metal complex (a–d) or free mAb (bottom panel). The cell lines were incubated with increasing concentration of mAb-metal complex at (a) 0.3; (b) 3; (c) 15, and (d) 40 pM; or with increasing concentration of mAb at (e) 0; (f) 10; (g) 55, and (h) 120 pM, respectively. The images were recorded either in emission intensity or lifetime. The scales of diagrams are 15 × 15 μm and the resolutions are 400 × 400 pixel with an integration of 0.6 ms/pixel. Bottom panel: (h) lifetime distributions over the entire images of blank cell and cell by the mAb-metal complex or labeled mAb in the absence of metal and (i) percentage distribution of occupation area by the mAb-metal complex throughout the entire cell image.