Research Article Nile Red and 2-NBDG Are Incompatible for the Simultaneous Detection of Lipid and Glucose Accumulation Andrew M. Hogan, Viswanathan Swaminathan, Nikitha K. Pallegar, and Sherri L. Christian Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, 232 Elizabeth Ave, St. John’s, NL, Canada A1B 3X9 Correspondence should be addressed to Sherri L. Christian; sherri.christian@mun.ca Received 14 October 2016; Revised 22 November 2016; Accepted 27 November 2016 Academic Editor: Jaan Laane Copyright © 2016 Andrew M. Hogan et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Glucose is the universal energy source and a critical substrate for lipid synthesis in mammalian cells. Analysis of both glucose and lipid in cells is important for the understanding of the regulation of lipid synthesis in many cell types, but especially adipocytes, the major storage cell for fat in mammals. Te fuorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of glucose, 2-NBDG, is used to monitor glucose uptake and the lipid-selective fuorophore Nile red is used to monitor lipid accumulation. Previous reports have used NBD-based fuorophores and Nile red simultaneously despite the possibility of spectral overlap. In this study, we determined if these fuorophores were experimentally compatible in preadipocytes and adipocytes stained with 2-NBDG and Nile red separately or costained. We found that Nile red is detectable in the wavelengths necessary to excite and detect 2-NBDG. Tis interference was further increased by the solvatochromic efect of lipid-localized Nile red. In addition, we found a synergistic increase in fuorescent intensity when both fuorophores were present. Unfortunately, even fne control of the excitation or emission wavelengths did not identify wavelengths suitable for selective detection when cells were costained. Terefore, 2-NBDG and Nile red cannot be used simultaneously—but can likely be used sequentially—to assess glucose uptake and lipid accumulation in lipid-laden cells. 1. Introduction Lipids accumulate in mammalian cells either through extrac- tion of dietary lipids from the circulation or by de novo lipogenesis using precursor molecules, of which glucose is a primary and essential substrate [1, 2]. While this process occurs in all mammalian cells, adipocytes are the predom- inant cellular constituent of adipose tissue where excess energy is stored as neutral lipids in the form of triacylglycerols and cholesteryl esters. Te study of lipogenesis as well as adipogenesis, which is the development of mature adipocytes from preadipocytes, has had a renewal in interest due to the rapid rise in obesity globally over the past 2 decades [3, 4]. Diferentiation of preadipocytes to mature adipocytes is characterized by the accumulation of lipid over the course of 1-2 weeks in vitro where the cell progresses through multiple stages characterized by key changes to gene expression [5]. Understanding the regulation of these processes is ofen dependent on measuring both lipid accumulation and cellular glucose uptake. A common cellular model used to study adipocyte biology is the 3T3-L1 cell line, which is murine embryonic preadipocytes capable of diferentiating in vitro into lipid-laden mature adipocytes [6, 7]. Te use of fuorescent glucose analogues has recently come into favour as a replacement for radiolabeled glucose for monitoring glucose uptake, following the general trend away from radioisotopes [8–10]. D-glucose is modifed by the covalent attachment of the 7-nitrobenz-2-oxa-1,3-diazolyl fuorophore to create 2-NBDG, a modifcation which has only minor efects on its uptake properties as, similar to D-glucose, uptake of 2-NBDG occurs through glucose transporters (GLUTs) and with comparable   values to D-glucose [11]. Typical excitation and emission wavelengths for detecting 2- NBDG (and other NBD-based fuorophores) are approxi- mately 480 nm and 550 nm, respectively [11, 12]. First applied over 30 years ago as a hydrophobic stain to visualize lipid droplets, Nile red is now recognized to stain neutral, polar, and charged lipids, and hydrophobic protein Hindawi Publishing Corporation International Journal of Spectroscopy Volume 2016, Article ID 5215086, 8 pages http://dx.doi.org/10.1155/2016/5215086