PAGE 5 OF 8 Nucleic Acids Research, 2005, Vol. 33, No. 22 e190 Figure 4. Sorting the effects of double gene knockdowns using two colors of QDs. (A) Schematic representation of cells transfected simultaneously with Lmna siRNA/green QD complexes and T-cad siRNA/orange QD complexes. The low 8% (L++, where ++ designates the presence of two colors of QDs) and high 8% (H++) of the dual fluorescence dot plot was gated and isolated using FACS. (B) Representative western blot and (C) corresponding band densitometry analysis of lamin A/C and T-cad protein levels in control unsorted (U) cells, unsorted (U) T-cad siRNA-treated cells, sorted T-cad/QD-treated cells (L+ and H+) and sorted dual siRNA/dual QD-treated (L++ and H++) cells. Consistent with the quantitative bulk protein assay, immuno- ﬂuorescent detection of lamin nuclear protein in unsorted, siRNA-transfected cells produced heterogeneous staining throughout the cell population (Figure 2A). However, in the co-transfected case, the presence of green QDs correlated with consistently weak lamin immunoﬂuorescent staining in the high co-transfected subpopulation (Figure 2B), compared to a lack of observable QDs and strong lamin staining in the low subpopulation (Figure 2C). Heterogeneous silencing therefore inﬂuences the accuracy of the bulk protein expression readout, suggesting the importance of verifying successful siRNA transfection for each gene knockdown study. Using QDs as photostable probes in combination with FACS, a subpopula- tion of uniformly-treated cells can be isolated, and also tracked with ﬂuorescence microscopy over long periods of time. This may be useful for observing the protein down-regulation and phenotypic responses of cells to gene regulation over time. To optimize the QD/siRNA correlative effect, we varied the ratio of QD to lipofection reagent with a ﬁxed dose of 100 nM siRNA. Speciﬁcally, we co-complexed Lmna siRNA with QD:lipofection reagent ratios of 1:5, 1:2, 1:1 or 2:1 (corres- ponding to 1, 2.5, 5 or 10 mg QD) and sorted the high 10% and low 10% of the cell ﬂuorescence distributions as before. We found that optimal ﬂuorescence and gene silencing correlation for the least amount of QD occurs at a 1:1 QD:lipofection reagent mass ratio (5 mg QD), as assayed by western blot (Figure 3A–C). We hypothesize that this optimum results from the limited surface area of the cationic liposome delivery agent ( 1 mm2) that is shared by the siRNA and QDs during the complexing process. Using too few QDs fails to provide ﬂuorescence that is detectable over background, whereas excess QDs occupy sites on the liposome that would otherwise be available to siRNA. In support of this theory, we found that saturating the liposome with QDs (100:1 ratio) before trans- fection abolished correlation between cellular ﬂuorescence and gene silencing; both high- and low-populations exhibited little to no knockdown (data not shown). QDs exhibit an extensive range of size- and composition- dependent optical properties, making them uniquely advantageous for multiplexing i.e. monitoring and sorting cells that have been treated simultaneously with different siRNA/QD complexes. As a proof-of-principle, we complexed cationic liposomes with either green (em 560 nm) QDs and Lmna siRNA or orange (em 600 nm) QDs and siRNA targeting T-cad. Cells were exposed simultaneously to both complexes and ﬂow cytometry was used to quantify orange ﬂuorescence (600 ± 10 nm) versus green ﬂuorescence (560 ± 20 nm) (Figure 4A). Cells exhibiting dual color ﬂuorescence were gated for low 8% and high 8% ﬂuorescence and collected. Western blots probing lamin a/c and T-cad protein conﬁrm speciﬁcity of QD/siRNA complexing (Figure 4B and C), while ﬂuorescence microscopy validates gating accuracy and demonstrates multi color tracking capabilities (Figure 5). Unsorted cells transfected with T-cad siRNA alone expressed a 45% down-regulation in protein expression quantiﬁed by Western blot band densitometry. In contrast, co-delivery of QDs with T-cad siRNA and subsequent sorting enabled sep- aration of the least efﬁciently transfected cell subpopulation