2006 Nature Publishing Group

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3 Supplementary Fig. 3: Supplementary EPR spectra Figure 1. X-band EPR differential spectrum obtained at 4.7 K after illumination (black line) of mercaptosuccinic acid (MSA) solubilized green QD. Frequency 9.4 GHz. Light source was 300 W Xe lamp, no cut off filter was used. The EPR spectrum is composed of four lines with a hyperfine coupling of a(h) α = 20 G and a(h) β = 13 G and corresponds to CHCH radical of MSA. The dotted line presents simulated spectrum g = Magnetic field (G) QD 560 QD Figure 2. X-band EPR differential spectra measured in dark at 4.7 K of yellow QD (grey line) and green QD (black line) Frequency 9.4 GHz. The solutions of dopamine conjugated QDs were exposed to air at ambient temperature for 2 hours. During exposure samples were protected from light. The signal with g = and H app = 16 G corresponds to the oxidized dopamine. In samples containing βme no signal due to oxidized dopamine was observed when exposed to air. 3400

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5 A 1 A 2 A 3 A 4 QD560 Only QD560 DA QD560 DAβME QD590 Only QD590 DA QD590 DAβME τ 1 τ 2 τ 3 τ A 1 τ 1 A 2 τ 2 A 3 τ 3 A 4 τ Σ i A i τ i Table 1. Best exponential fits to time-resolved data of two colours of QDs, green (560) and yellow (590), either unconjugated (QD only) or conjugated to dopamine (QDDA) or conjugated to dopamine and in the presence of 5.7 mm βme (DAβME). The relative amplitudes (A) and time constants in ns (τ) are given. The values A i τ i give a rough estimate of areas under the curve, and thus the relative amount of steady-state fluorescence likely to be contributed by this component. The sum over all of the components is given in the final row, and should give a rough estimate of the overall fluorescence of the QDs. The fits are plotted vs. the raw data in Fig. 2b. All numbers are rounded to three significant figures after calculating values.

6 Supplementary Methods Preparation of conjugates. Concentrations of dopamine used in the conjugates was 10 mm; of QDs, µm. The ph of all solutions was adjusted to neutral ( ). After conjugation, QDs were dialyzed vs. ddh 2 O to remove unbound dopamine. QDs were characterized before and after conjugation using TEM and a particle sizer (Malvern). Aggregates after conjugation were identified as a peak of >25 nm hydrodynamic radius on the particle sizer, and were removed by centrifugation before addition to cells. Controls and time course. Controls for all labelling experiments included solubilized QDs without conjugate; QDs with dopamine but no EDC; and dopamine alone without QDs (final concentration on cells, 1-10 mm). Stern-Volmer plots were obtained by adding aliquots of a 10 mm dopamine stock solution to optical-density-matched QD solutions. For time course experiments under room air and room light, conjugates were removed from the glove box and placed into open wells of a 96-well plate. When UV exposure was desired, a handheld UV wand on the long wavelength setting (365 nm) was held over the plate. For time course experiments on a slide, both with and without cells, an image cube was taken with the minimal exposure required to obtain the full peak ( ms, depending upon the magnification and filter used), and the shutter was then closed. This procedure was repeated until a total of s of data were obtained. The brightness of QDs on a slide was estimated from a (10 µm) 2 area that contained no visible aggregates. Absorbance readings. To determine the number of oxidized dopamine molecules in solution and/or adhered to QDs, QD-dopamine conjugates were prepared at twice the usual concentration and, after purification by dialysis, further concentrated using a Nanosep 10,000 molecular weight spin column until the optical density at the exciton peak was Unoxidized dopamine could not be resolved at these concentrations, but oxidized dopamine peaks at 420 nm could be seen in all conjugates. After concentration, the sample was divided into two wells of a quartz absorbance plate. The absorbance spectrum was measured, then both wells were exposed to a UV wand for the designated amount of time. Another reading was taken, then one of the samples was purified in a Nanosep column to remove unbound dopamine; this sample yielded the bound values. The other sample was not further purified and yielded the free values, representing all of the dopamine molecules that were adhering to the QDs after the initial EDC conjugation and dialysis. These two samples provided a measure of both oxidation rates and cap decay. Cell lines and labelling. Mouse epithelial A9 cells expressing the human D2 dopamine receptor were purchased from ATCC (ATCC CRL-10225). Negative controls included human embryonic kidney (HEK) 293 cells and mouse 3T3 fibroblasts. Cells were maintained in high glucose Dulbecco s Modified Eagle s Medium (DMEM) supplemented with 10% fetal calf serum, 0.2 mm glutamine, 100 U/mL penicillin and 100 µg/ml streptomycin in a 5% CO2 atmosphere at 37 C. For passage, cells were rinsed first with phosphate-buffered

7 saline (PBS) and then with Hanks balanced salt solution containing 0.05% trypsin and 0.02% EDTA, incubated for 2 min at room temperature, and resuspended in supplemented DMEM. For labelling with QD-dopamine, growth medium was removed by 2 washes in sterile PBS, and then replaced with 1 ml serum-free medium without phenol red (OptiMem, Gibco). 0.1 ml of the QD-dopamine conjugate was then added, the dishes were swirled for 30 s, then returned to the incubator for a 1 h incubation at 37 ºC in the dark. Before imaging, the cells were rinsed twice and imaged in PBS. For studies of effects of fixation, cells were fixed in 3% paraformaldehyde in PBS for 30 min, rinsed twice in PBS, and imaged in PBS. Control experiments were performed without cells: in this case, QD conjugates were added to OptiMem in the above concentrations and incubated in the tissue culture incubator for designated amounts of time. Spectra from these controls were collected on the plate reader. When used, β-mercaptoethanol was added to cells at the same time as the QDs, to a final concentration of 5.7 mm. Controls were performed substituting 5.7 mm mercaptoacetic acid for mercaptoethanol, to control for solubility effects of thiol compounds; these controls were performed three times for each colour of QD and abandoned when no increased solubility was seen. Ascorbic acid was also used as an antioxidant, as a freshly prepared solution at a concentration of 20 mm. Similar effects on cell survival were seen when ascorbic acid was used as an antioxidant instead of βme (not shown; n = 4 trials). However, QDdopamine fluorescence was weaker under these conditions and thus the use of ascorbic acid was not pursued. Assays for cell membrane damage and oxidative damage To quantify cell membrane damage, the cell-impermeant nucleic acid stain SYTOX Green (Molecular Probes) was added to cells after QD incubation. The final concentration used was 1 µm and the incubation time was min. SYTOX Green was visualized with an Endow GFP filter set (excitation = 470/40 nm, dichroic 495 nm, emission = 525/50; Chroma filter 41017) and its spectrum was confirmed using the multispectral imaging system. Numbers of live vs. dead cells were estimated by counting >100 cells in several low-power fields. Control dishes contained 3 ± 2% SYTOXpositive cells (n = 5 dishes); in labelled dishes, < 5% positive was taken as not significant. For studies of phototoxicity, SYTOX Green was added to cells after illumination under a Hg lamp. In this case, irradiated areas could be readily identified after staining by the relative brightness of the QD labelling as compared with unirradiated areas. The number of SYTOX-positive cells was then compared in irradiated vs. unirradiated regions of the same dish. Dihydroethidium (DHE) was also used to indicate reactive oxygen species and thus the potential for oxidative damage. Actively proliferating control dishes contained 20 ± 5 %

8 DHE-positive cells (n = 5 dishes). A positive result in a labelled culture was considered to be 100% DHE-positive cells. Oxidative DNA damage was quantified from genomic DNA purified from cultures using a DNEasy extraction kit (Qiagen). DNA was at 100 ng/ml in H2O with an absorbance ratio A260/A280 > 1.8. Oxidative damage was assayed using an oxidative DNA damage kit (quantitative) (Kamiya Biomedical), which provides a colourimetric measure of abasic sites that are calibrated against a standard curve. Establishment of QD quenching by RedoxSensorRed (RSR) Quenching of QD-fluorescence by RedoxSensor Red was tested under Hg lamp illumination in order to unquench the QD-dopamine conjugates. Varying amounts of dye (0.1 5 µm) were added to a given amount of QD-dopamine and fluorescence monitored under illumination for 2-5 min. The dye was considered to quench QD fluorescence if the QD emission peak remained more than five-fold weaker than an exposed spot containing QD-dopamine without dye. ICP-MS Cadmium concentrations in the supernatant of centrifuged solutions were determined using ion chromatography/mass spectrometry (ICP-MS). QD conjugates were tested after storage in the dark under Ar, or after 2 h of exposure to a UV wand (365 nm) and room air. Controls were prepared containing 1 mg/ml EDC and 2 mm dopamine in PBS to control for possible chemical contamination. We used a PerkinElmer/SciEx Elan DRCplus equipped with a Meinhard nebulizer and a cyclonic spray chamber, all located in a HEPA-filtered environment. Several standard solutions bracketing the sample concentrations were prepared from SCP brand standard solutions and >18 MΩ deionized H 2 O. The source of H 2 O was the same as was used for all QD and conjugate preparation. Both standards and samples were run in one percent nitric acid solutions using indium as an internal standard. Samples were diluted 100-fold to reduce organic loading and reduce signal intensities. Cadmium blanks were on the order of 3 ppt and were subtracted from both the standard and sample intensities. Reported sample concentrations are precise to 5 % relative. EPR EPR experiments were conducted on a Bruker ESP300E spectrometer equipped with a Varian cavity and a variable-temperature cryostat (Air Products) cooled to liquid helium temperature. The microwave frequency was determined after each measurement using a Hewlett-Packard 5352B frequency counter. The g tensors were calibrated for homogeneity and accuracy by comparison to a coal standard, g = ± Samples were excited directly into the cavity using a 300-W Xe lamp (ILC) with a 355 nm cutoff filter. The filter was used to avoid possible excitation of dopamine. Samples were checked for background EPR signals before irradiation.