e190 Nucleic Acids Research, 2005, Vol. 33, No. 22 PAGE 4 OF 8 Figure 2. Immunofluorescence staining of Lamin A/C nuclear protein (red). (A) Unsorted cells (U) transfected with Lmna siRNA alone display heterogenous staining for nuclear lamin throughout the cell population. White arrows highlight examples of cells with weak lamin staining among cells stained strongly for lamin. (B) Cells co-transfected with Lmna siRNA and green QDs exhibit bright lamin staining and lack of QDs in low-gated (L+) cell subpopulations and (C) weak lamin staining and presence of QDs in high-gated (H+) cell subpopulations (shown enlarged in inset). Scale bars 75 mm. penicillin and 10 mg/ml streptomycin. After 24 h of hepatocyte seeding, the ﬁbroblasts from transfection experiments were co- cultivated at a previously optimized 1:1 hepatocyte:ﬁbroblast ratio in ﬁbroblast medium (36). Medium from hepatocyte/ ﬁbroblast co-cultures was collected and replaced with hepato- cyte medium every 24 h until completion of the experiment. Hepatocellular function assays Hepatocyte/ﬁbroblast co-cultures were assayed for albumin production and cytochrome P450 enzymatic activity, proto- typic indicators of hepatocellular function (37,38). Albumin content in spent media samples was measured using an enzyme linked immunosorbent assay (ELISA) with HRP detection (35). Cytochrome P450 (CYP1A1) enzymatic activ- ity was measured by quantifying the amount of resoruﬁn produced from the CYP-mediated cleavage of ethoxyresoruﬁn O-deethylase (EROD) (39). Speciﬁcally, EROD was incub- ated with cell cultures for 30 min, media was collected and resoruﬁn ﬂuorescence quantiﬁed at 571/585 nm excitation/ emission. Error bars represent standard error of the mean (n ¼ 3). Statistical signiﬁcance was determined using one-way ANOVA. RESULTS AND DISCUSSION We used cationic liposomes to co-deliver green QDs and siRNA targeting the lamin a/c gene (Lmna) into murine ﬁbro- blasts, followed by ﬂow cytometry to quantify intracellular QD uptake (Figure 1A). The median ﬂuorescence of QD/ siRNA-transfected cells compared to mock-transfected cells (liposome reagent only) and cells transfected with siRNA alone varied by 84% (coefﬁcient of variation). FACS was used to gate and collect the brightest 10% (high, H) of each ﬂuorescence distribution, along with the dimmest 10% (low, L). After the sorted cells were re-plated and grown for 72 h to ensure protein turnover, protein expression analysis by either western blot or immunostaining was performed. In cells that had been co-transfected with siRNA and QDs, gene silencing correlated directly with intracellular ﬂuorescence. Western blotting (Figure 1B) and image analysis of lamin a/c protein bands (Figure 1C) show 90% knockdown in the highly ﬂuor- escent cells and negligible knockdown in the dimmest cells. The cells treated with siRNA alone exhibited mediocre gene down-regulation (20–30%) independent of sorting parameters. Figure 3. Optimization of QD concentration for siRNA tracking. Lmna siRNA (100 nM) and 1, 2.5, 5 or 10 mg QD were co-transfected into murine fibroblasts and the cells FACS-sorted for the low 10% (L+) and high 10% (H+) of intra- cellular fluorescence distribution. (A) Protein expression of sorted cells assayed by western blot, b-actin loading control. Unsorted, lipofectamine only control (U) represented 100% lamin a/c protein expression. (B) Western blot band densitometry analysis of L+ and H+ bands shows an optimum QD concentration for obtaining high-efficiency silencing. (C) Band density difference (L+ minus H+) reveals an optimum QD concentration for sorting most efficiently silenced from least efficiently silenced subpopulations.