microscopic examination, live animal and tissue section imaging studies [15]. Recently, a toxicity study on IRDye 800CW revealed that there was no observed adverse effect at a dose of approximately 10,000 times higher than the projected dose for in vivo imaging. This is the first toxicity study on a NIR dye with the functional labeling potential [16]. In this study, the BG-800 substrate was synthesized by a one- step reaction between IRDye 800CW-NHS ester and BG-NH2. BG-800 was characterized using both cell-based assay and in vivo imaging. To reduce the background, quenched substrates containing IRDye 800CW and IRDye QC1 conjugated at the benzyl and guanine groups of BG, respectively, were created and characterized. Materials and Methods Ethics statement All experimental procedures for the use of animals were previously reviewed and approved by the institutional animal care and use committee (IACUC) at the University of Nebraska- Lincoln (protocol #402), and all of the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Chemicals and reagents The SNAPf-Beta-2 adrenergic receptor (SNAPf-ADRb2) vector, amine-terminated building block BG-NH2, BG-782 (SNAP- Surface 782) and purified SNAPf-EGF protein were from New England Biolabs (Ipswich, MA). The IRDye 800CW and IRDye QC1 were from LI-COR Biosciences (Lincoln, NE). The synthesis of BG substrates (BG-800, CBG-800-QC1 and CBG-800-PEG- QC1) was conducted at New England Biolabs based on previously published methods [10]. The structures of these substrates are shown in Fig. S1. All substrates were reconstituted in DMSO to 1 mM as stock solutions. TO-PRO-3 and DAPI nucleus staining reagents were purchased from Invitrogen (Carlsbad, CA). Cell culture The human ovarian adenocarcinoma cell line SK-OV-3, breast adenocarcinoma cell line MDA-MB-231 and embryonic kidney 293 (HEK293) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HEK293 and MDA- MB-231 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FBS) and 1% penicillin-streptomycin (complete DMEM). SK-OV-3 cells were maintained in McCoy’s 5 A medium (McCoy) supplemented with 10% fetal calf serum (FBS) and 1% penicillin-streptomycin (complete McCoy). To collect cell lysates, cells were rinsed with PBS once before adding RIPA buffer. The cells were then incubated on ice in RIPA buffer for 15 min. The cell lysates were collected and centrifuged at 4uC to separate the supernatant from the cell debris. Cell transfection and stable cell line generation The cell transfection procedure was modified from the previously described method [17]. Lipofectamine 2000 (Invitro- gen, Carlsbad, CA) was used to deliver the SNAPf-ADRb2 plasmid into the cells. Two days after transfection the cells could be used for in vitro imaging studies as described below. For stable cell selection, the culture medium was replaced with that contains G418 at 24 h after transfection. The G418 resistant cells were pooled and stored for future use. NIR Fluorescence Imaging with SNAP-Tag Reaction of BG substrates with cell lysate BG substrates were added to cell lysate (1 mg/ml) to a final concentration of 500 nM. The reaction was conducted at room temperature. The reaction mixtures were resolved by gel electrophoresis. The labeled protein bands were visualized by scanning the gel on an Odyssey Infrared Imaging System (LI- COR Biosciences). Cell staining with BG substrates in 96-well plates Cells were seeded at approximately 36104 (MDA-MB-231), 26104 (HEK293) or 16104 (SK-OV-3) cells per well in 96-well plates and cultured overnight before the assay. The cell density was about 80–90% confluent at the time of assay. The BG substrates were diluted in complete cell culture medium to designated concentrations and incubated in the 37uC 5% CO2 incubator for 30 min except where otherwise stated. Cells were fixed with 3.7% formaldehyde/PBS and washed with PBS containing 0.1% Tween 20 (PBST), and then incubated in TO- PRO-3 nucleus stain agent (1:5000 in PBS) to normalize for cell numbers. After three additional washes with PBST, the plates were scanned and signal intensity quantified [18]. Microscopic analysis Cells were seeded in 6-well plate with cover slips and cultured overnight. The cells were incubated with 1 mM BG substrates at 37uC 5% CO2 for 30 min, fixed, permeabilized and washed as described above. Instead of TO-PRO-3, DAPI nucleus staining agent was used to visualize the nuclei by microscopy. After the final wash, the cover slips were mounted on glass slides with Fluoromount reagent (Sigma, St. Louis, MO). The images were acquired using an Olympus IX81 Inverted microscope system equipped with a halogen bulb (Olympus, Hamburg, Germany). NIR filters (EX: 710/75 nm, EM: 810/90 nm; Chroma Tech- nology Corp., Rockingham, VT) were used for IRDye 800CW detection. The images were deconvolved using the accompanying software. Xenograft mouse model Mice were maintained on a purified maintenance diet (AIN- 93M) from Harlan Teklad (Madison, WI). The xenograft tumors were established as previously described with modifications [19]. In brief, athymic nude (nu/nu) mice, obtained from Charles River Laboratories, Inc. (Cambridge, MA) at 4 weeks of age, were subcutaneously injected with 56106 SK-OV-3 cell suspension in 0.1 ml serum-free media. Imaging studies began when tumors reached about 5 mm in size. In vivo animal imaging Mice were anesthetized with 2% isoflurane throughout the procedures. For imaging experiments, the BG substrates were diluted in 100 ml PBS and injected through the tail vein. The images were acquired at indicated time points with a Pearl Impulse Imager (LI-COR Biosciences). The Ex/Em settings for the 700 nm channel and 800 nm channel were 685/720 nm and 785/820 nm, respectively. The images were analyzed using the accompanying software [14]. Organ and tissue analysis Mice were sacrificed at 1 d after BG substrates injection, and dissected to collect the organs. The excised organs were rinsed in PBS, and imaged using a Pearl Impulse Imager. For gel analysis, tissue samples were homogenized in RIPA buffer. After centrifu- PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34003