Quantum Dot – Antibody Conjugates for Labeling Neutrophils

Abstract

Abstract: : Purpose: Leukocytes have a significant role in inflammatory processes in the retina. Current methods of studying leukocyte trafficking within the microcirculation would be enhanced by fluorescent labeling which is more photostable, with features of high quantum efficiency and greater cell type specificity. Our goal is to develop an in vivo leukocyte imaging probe that incorporates these features, for the purpose of studying leukocyte trafficking in detail. As a first step, we tested a technique for specifically labeling neutrophils with quantum dot – antibody conjugates ex vivo, and observed these cells in the rat retinal circulation. Methods: Rat blood cells were separated by Percoll density gradient centrifugation. Following isolation and resuspension, neutrophils, lymphocytes, and monocytes were incubated with a mouse anti–rat neutrophil antibody (Pharmingen) – quantum dot conjugate. Unbound conjugate was rinsed thoroughly from the cells. A microplate reader was used to observe intensity photostability, and specificity of the conjugates in vitro. Specificity of neutrophil targeting was also assessed by fluorescence microscopy. Neutrophils were resuspended in a saline medium, and reinfused into the rat by tail vein catheterization. Retinal real–time image acquisition was performed using an inverted fluorescence microscope during reinfusion. Comparative studies were performed with conventional methods using the same technique with Percoll–isolated cells labeled with acridine orange ex vivo. Results: Microplate reader analysis indicated significantly more fluorescence in the neutrophil cell fraction compared to monocyte and lymphocyte fractions, suggesting cell specificity. In addition, time series microplate analysis confirmed the increased photostability of the quantum dot conjugate relative to acridine orange. Fluorescence microscopy revealed specific neutrophil cell surface labeling by the quantum dot–antibody conjugate. In vivo analysis indicated that quantum dot–labeled cells were intense and photostable, and could be tracked throughout the retina. Conclusions: We have demonstrated that in vivo imaging of quantum dot–labeled neutrophils is a potentially useful method for observing neutrophils in the circulation. Photostability, intensity, and specificity of cell binding are advantages of this technique which could be applied to studies of various cell types in vivo.

Keywords: imaging/image analysis: non-clinical • retina • inflammation