gation, the supernatants were run on a SDS-PAGE gel and analyzed by in-gel fluorescence scanning. Results BG-800 labeling of cells transiently transfected with SNAPf-ADRb2 A SNAPf-ADRb2 expressing plasmid was used in this study. The ADRb2 portion of the fusion protein directs the localization of the protein to the cell membrane. HEK293 cells were transiently transfected with SNAPf-ADRb2 using a Lipofectamine 2000-mediated method. After 2 d culture, the transfected cells were labeled with BG-800. Because the labeling is irreversible, excess substrate could be washed away [12]. The fluorescence signal on SNAPf-ADRb2 transfected cells was about 24 times higher than that of un-transfected HEK293 cells (Fig. 1), indicating that the BG-800 substrate could react with SNAPf- ADRb2 protein. Microscopic examination revealed that the fluorescence signal was mainly on the cell membrane (Fig. 1, inset). Generation of stable cells expressing SNAPf-ADRb2 and labeling with BG-800 HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAPf-ADRb2 were selected using G418-containing medium. These cells were designated as 293-SNAPf, MDA-SNAPf and SKOV-SNAPf, respectively. The expression of SNAPf-ADRb2 in the stable cells was determined by the reaction between BG-800 and cell lysates. Fig. 2A shows a representative gel image in which the reaction mixtures were resolved. Each of the three stable cell lines showed a positive band, presumably resulting from the reaction between SNAPf-ADRb2 and BG-800. As a comparison, this band was absent for parental cell lines. The calculated molecular weight of SNAPf-ADRb2 is about 70-kDa, which matches the molecular weight of the bands on the gel. Microscopic examination also confirmed the SNAPf-ADRb2 expression in the stable cells. It also demonstrated that the fluorescence signal was predominantly on the cell membrane (Fig. 2B), indicating the correct localization of SNAPf-ADRb2 Figure 1. Reaction of BG-800 with HEK293 cells expressing SNAPf-ADRb2 protein. HEK293 was transiently transfected with SNAPf-ADRb2 expression plasmid. After 2 d of culture, cells were reacted with BG-800. The fluorescence signal on the cells was measured by scanning on Odyssey Infrared Imaging System. HEK293 cells transfected with empty vector were used as a control. Shown in the inset is a representative microscopic image of BG-800 stained HEK293 cells expressing SNAPf-ADRb2. doi:10.1371/journal.pone.0034003.g001 NIR Fluorescence Imaging with SNAP-Tag Figure 2. Confirmation of SNAPf-ADRb2 stable cell lines. (A) HEK293, MDA-MB-231, and SK-OV-3 cells stably expressing SNAPf- ADRb2 (designated as 293-SNAPf, MDA-SNAPf and SKOV-SNAPf, respectively) were lysed in RIPA buffer. The cell lysates were reacted with BG-800 substrate and run on a SDS-PAGE gel. The 70-kDa bands represented the SNAPf-ADRb2 protein. The parental cell lines of the stable cells were used as negative controls. 1, HEK293; 2, 293-SNAPf; 3, MDA-MB-231; 4, MDA-SNAPf; 5, SK-OV-3; 6, SKOV-SNAPf. (B) Microscopic examination of BG-800 reaction with stable cell lines expressing SNAPf- ADRb2. 293-SNAPf, MDA-SNAPf and SKOV-SNAPf cells were incubated with BG-800 in complete culture medium. The cells were then fixed and stained with DAPI to show the nuclei. The parental cells of each cell lines were used as negative controls. Note that BG-800 reaction signals were mainly on the cell membrane. Scale bar: 10 mm. doi:10.1371/journal.pone.0034003.g002 protein. However, signals were also observed inside the cells, probably representing internalized SNAPf-ADRb2 after labeling. The reaction signal is dependent on BG-800 concentration The reaction signals between BG-800 and SNAPf-ADRb2 stable cells were dependent on BG-800 concentration. With the increase of BG-800 concentration, the signal intensity also increased (Fig. 3). All three cell lines showed a similar trend, and the maximum signals were reached when approximately 200 nM PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34003