Eight well chambered cover glasses

Mouse primary cortical neurons were cultured on eight-well chambered cover glasses (Nalge Nunc International, Rochester, NY). After incubation with FAM-Aβ42 (500 nM) at 37 °C for 6 h, together with or without apoE3 particles (200 nM) in the presence or absence of heparin (15 U/ml), fluorescence associated with Aβ was observed by confocal laser-scanning fluorescence microscopy (model LSM510 invert; Carl Zeiss, Jena, Germany). LysoTracker (50 nM; Invitrogen) was added to determine lysosome-Aβ colocalization 30 min before confocal imaging.

Cells were grown on eight-well chambered coverglasses (Nunc, Langenselbold, Germany) overnight (37 °C, 96% humidity and 5% CO₂). After washing with PBS, cell membranes were stained with 0.25 µg/mL CellMask^TM DeepRed (Invitrogen) in PBS for 5 min and washed twice with PBS. For observing NP uptake kinetics, cells were incubated with PBS containing NPs at specified concentrations (QDs, 10 nM; anionic and CPS, 75 µg/mL; cationic and NPS, 7.5 µg/mL). AuNCs were suspended in serum-free DMEM at 20 µg/mL. Live cell imaging was performed for up to 2 h with our spinning disk laser scanning confocal microscopy systems [29 (link),31 (link)]. Images were acquired in two separate color channels. NP emission was collected through a bandpass filter (AHF, Tübingen, Germany). Detailed information on the experimental conditions is given in Table 3. Suitable control experiments were performed to ensure negligible crosstalk between the two channels. Images were quantitatively analyzed using the software ImageJ [51 ]. The fluorescence intensity of NPs in each cell was obtained by normalizing the integrated intensity by the cell area. The cell membrane and the intracellular region were identified based on the membrane staining.

Eight-well chambered coverglasses (Nunc, Rochester, NY) were prepared by plasma cleaning followed by incubation over night with polylysine-conjugated polyethylene glycol (PEG-PLL), prepared using a modified Pierce PEGylation protocol (Pierce, Rockford, IL). PEG-PLL coated Chambers were rinsed with and stored in Milli-Q water until use. FCS measurements were done on a lab-built instrument based on an Olympus IX71 microscope with a continuous emission 488 nm DPSS 50 mW laser (Spectra-Physics, Santa Clara, CA). All measurements were done at 20 °C. The laser power entering the microscope was adjusted to 4.5 μW. Fluorescence emission collected through the objective was separated from the excitation signal through a Z488rdc long pass dichroic and an HQ600/200 m bandpass filter (Chroma, Bellows Falls, VT). Emission signal was focused through a 50 μm optical fiber. Signal was amplified by an avalanche photodiode (Perkin Elmer, Waltham, MA) coupled to the fiber. A digital autocorrelator (Flex03Q-12, correlator.com, Bridgewater, NJ) was used to collect 30 autocorrelation curves of 30 seconds for each measurement. Fitting was done using MATLAB (The MathWorks, Natick, MA).