Abstract 214: Characterization of the near-infrared fluorescent protein iRFP for plate-based assays and animal imaging

Abstract

Abstract Fluorescence imaging has been extensively used in biological research because of its high sensitivity, signal stability, and simplicity of multiplexing. Compared to fluorophores in the visible spectral region, fluorophores with excitation (Ex) and emission (Em) maxima in the near-infrared (NIR) region (700-900 nm) are desirable in many applications. Cells, buffers and plastic materials generate less background fluorescence in the NIR region. As a result, NIR fluorescence imaging offers higher sensitivity and better signal-to-background ratio (SBR) compared to visible fluorescence. In addition, due to the reduced light absorption and scattering in animal tissues, NIR fluorescence is particularly suitable for in vivo imaging studies. In this study, we characterized the NIR fluorescent protein iRFP for plate-based assays and animal imaging. HEK293 cells stably expressing iRFP were established and designated as HEK293-iRFP. Serial dilutions of HEK293-iRFP cells were seeded in 96-well plate and scanned on the Odyssey CLx Imaging System. A good linear relationship between fluorescence signal intensity and cell number was obtained. The lower limit-of-detection was determined as 763 cells. To evaluate the performance of iRFP for animal imaging, the iRFP-expressing plasmid was delivered into mouse liver cells using a hydrodynamic injection method. A dose- and time-dependent pattern of fluorescence signal was observed after plasmid injection. At 8 h post-injection of 10 μg of iRFP plasmid, a SBR of 41 was observed by live animal imaging. Analysis of liver tissue lysates revealed a SBR of 148. Other tissues, including kidney, spleen, heart and lung, showed very little fluorescence signal. Our results demonstrate that iRFP is a good reporter for both plate-based assays and animal imaging studies. Citation Format: Haibiao Gong, Joy L. Kovar, Teresa Urlacher. Characterization of the near-infrared fluorescent protein iRFP for plate-based assays and animal imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 214. doi:10.1158/1538-7445.AM2015-214