e190 Nucleic Acids Research, 2005, Vol. 33, No. 22 the limited selection of available reporters currently prevent RNAi tracking from being feasible in either long-term or multiplexed studies. The dyes commonly used to label siRNAs lose over half the intensity of ﬂuorescent signal in 5–10 s (22,23). Meanwhile, ﬂuorescent reporter plasmids, although meant to be continuously expressed by the cells, can require as long as 2 h after transcription for the functional protein to be observable (24). Perhaps most importantly, due to the limited availability of ﬂuorophores and reporter proteins that have non-overlapping emission spectra, current transfection screen- ing methods are incapable of simultaneous monitoring of multiple siRNA molecules. In this study, we demonstrate a novel means of monitoring nucleic acid delivery using standard transfection techniques to co-deliver semiconductor quantum dots (QDs) along with siRNA. QDs are bright, photostable CdSe/ZnS ﬂuorescent nanocrystals that exhibit tunable emission properties for a wide range of color possibilities. We and others have shown that QDs can be rendered non-cytotoxic (25) and innocuous to normal cell physiology and common cellular assays, such as immunostaining and reporter gene expression (26). Combin- ing QDs with siRNA for RNAi tracking requires neither chem- ical labeling of siRNA, which is costly and can potentially deter complexing with RISC, nor the expression of reporter plasmids. QDs are also brighter than most conventional ﬂuor- escent dyes by 10-fold (22,27) and have been signiﬁcantly easier to detect than green ﬂuorescent protein (GFP) among background autoﬂuorescence in vivo (27). Furthermore, QDs are far less susceptible to photobleaching, ﬂuorescing more than 20 times longer than conventional ﬂuorescent dyes under continuous mercury lamp exposure (28). Using our QD/siRNA co-delivery technique, we found that cellular ﬂuorescence correlated with level of silencing, allowing collection of a uniformly silenced cell population by ﬂuorescence-activated cell sorting (FACS). The superior brightness and photostabil- ity of these probes in cells sustained not only FACS, but also live imaging, and immunostaining procedures. With two QD colors and two siRNAs as a model, we also demonstrate the method’s unique ability to generate cell populations with multiplexed levels of knockdown. Finally, we show that a homogenous silenced cell population generated using this method is essential to observe the phenotypic effects of decreased T-cadherin (T-cad) protein expression on cell–cell communication between hepatocytes and non-parenchymal cells. MATERIALS AND METHODS Short interfering RNA and QD preparation Pre-designed siRNA was used to selectively silence the Lamin A/C gene (Lmna siRNA #73605, NM_019390, Ambion) and the T-cad gene (SMARTpool reagent CDH13, NM_019707, Dharmacon). Fluorescently-labeled Lmna siRNA purchased from Dharmacon was designed with a ﬂuorescein molecule on the 50 end of the sense strand. The annealed sequences were reconstituted in nuclease-free water and used at a concentra- tion of 100 nM (Lmna siRNA, 50-Fluorescein-Lmna siRNA) or 50 nM (T-cad siRNA). Green (560 nm emission maxima) and orange (600 nm emission maxima) CdSe-core, ZnS-shell nanocrystals were PAGE 2 OF 8 synthesized and water-solubilized with mercaptoacetic acid (MAA) as described previously (29–31). MAA-QDs were then surface-modiﬁed by reacting with polyethylene glycol (PEG)-thiol MW 5000 (Nektar) overnight at room temperat- ure. Excess PEG-thiol was removed by spin ﬁltration (100 kDa cut-off). QDs are also available commercially as an alternative to synthesis (Quantum Dot Corp., Evident Technologies). Unless stated otherwise, 5 mg PEGylated QD was used per cell transfection. Fibroblast cell culture and transfection Fibroblasts (3T3-J2) were provided by Howard Green (Harvard Medical School, Cambridge, MA) (32) and cultured at 37 C, 5% CO2 in DMEM with high glucose, 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The transfection procedure was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruc- tions. Brieﬂy, 3T3 ﬁbroblasts were plated 24 h before trans- fection at a density of 3 · 106 cells per 35 mm well, in antibiotic- and serum-free medium. Lipofectamine reagent (5 ml) and either siRNA or QDs were diluted in DMEM and complexed at room temperature. For QD/siRNA co- complexes, siRNA and liposomes were allowed to complex for 15 min before an additional 15 min incubation with QDs. Complexes were added to cell cultures in fresh antibiotic- and serum-free medium until 5 h later, at which time the cultures were washed and replaced with regular growth medium. Approximately 24 h post-transfection, cells were trypsinized and prepared for ﬂow cytometry. FACS Flow cytometry and sorting was performed on a FACS Vantage SE ﬂow cytometer (Becton Dickinson) using a 488 nm Ar laser and FL1 bandpass emission (530 ± 20 nm) for the green QDs, FL3 bandpass emission (610 ± 10 nm) for the orange QDs. Fluorescence histograms and dot plots were generated using Cell Quest software (for ﬁgures, histograms were re-created using WinMDI software, http://facs.scripps. edu). Cell Quest was also used to gate populations of highest and lowest ﬂuorescence intensity for sorting into chilled FBS. Sorted populations were immediately re-plated into separate wells containing regular growth medium and allowed to adhere. Cells were incubated at 37 C until visualized by ﬂuor- escence microscopy or until assayed for protein level. Western blotting Cell cultures were scraped and lysed in RIPA Lysis Buffer (Upstate Biotechnologies) supplemented with COMPLETE EDTA-free Protease inhibitor solution (Roche). Equal amounts (15–20 mg) of total protein were loaded into 10% Tris–HCl resolving gel, separated by electrophoresis and transferred to PVDF membrane. The blot was incubated in blocking solution (5% [w/v] non-fat dry milk, 200 mM Tris base (pH 7.4), 5 M NaCl and 5% Tween-20) for 1 h at room temperature, primary antibody overnight at 4 C, and second- ary antibody for 1 h. Three washes in 200 mM Tris base (pH 7.4), 5 M NaCl and 5% Tween-20 took place between steps and after completion of probing. Finally, the blot was visualized by chemiluminscence (Super Signal West Pico Kit, Pierce) and developed. Bands were analyzed for density using