SH-SY5Y neuroblastoma cells (ATCC) were kept in DMEM:F12 (1:1) supplemented with 2 mM glutamine, 1% non essential amino acids and 15% fetal bovine serum (FBS). Cell exposure to particles Cells (at density of 0.36106/ml for RAW 264.7 macrophages and HeLa cells, 0.56106/ml for HMDM and MDDC, 36105/ml for microglia and SH-SY5Y neuroblastoma cells) were exposed to non-coated SWCNT or PC-coated SWCNT, PS-coated SWCNT (0.02 mg/ml, calculated by concentration of SWCNT, or 0.1 mg/ ml for HMDM and MDDC) for 2 h (and 24 h for some experiments using MDDC) in serum-free medium, except for primary human phagocytes which were maintained in cell culture medium supplemented with 10% heat-inactivated serum so that cell viability was not compromised. For the purpose of uniformity we present the condition of exposure to SWCNT using the ratios of SWCNT (mg)/106 cells. The ratios SWCNT to cells were 125– 150 mg/106 cells. In the experiments with cyt c/PS-coated SWCNT chloroquine (100 mM) was applied as endosome disruptor and cells were incubated for 15 min at 37uC. Cells were washed once with DMEM medium and then incubated in DMEM medium containing 10% FBS for additional 2 h. At the end pointed incubation, cells were washed with PBS, collected and used for assessment of caspase 3/7 activity and externalized of PS. Cytotoxicity was confirmed using Trypan blue exclusion as well as LDH release (with CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega, WI) kit). Assessment of cytokines RAW macrophages were seeded at 2.56105 cells/well 12 h before treatment. Cells were incubated with 0.25 mg/ml zymosan in the presence or absence of SWCNT, PS-coated SWCNT or PC-coated SWCNT (all at the level of 150 mg/106 cells) in normal culture medium for different time periods. At the end of incubation, the medium was collected and subjected to 50,000 g centrifugation at 4uC for 30 min to remove nanoparticles from the solution. The supernatant was then used to measure cytokines. R&D Quantikine H immunoassay kit (R&D system Inc. Minnea- polis, MN) was used for measurements according to the manufacturer’s manual. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay kit (Promega, Madison WI, USA). PS exposure was determined by fluorescently microscopic detection of annexin V as outlined in the annexin V-FITC apoptosis detection kit (BioVision Research Products, Mountain View, CA). Cells were analyzed under a Nikon ECLIPSE TE 200 fluorescence microscope (Tokyo, Japan) equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR) using the MetaI- maging SeriesTM software version 4.6 (Universal Imaging Corp., Downingtown, PA). A minimum of 300 cells were analyzed per experimental condition. Flow cytometry Fluorescence intensity of HMDM or MDDC incubated in the presence or absence of NBD-labeled PC/PS-coated SWCNT was measured with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser. Ten thousand events were gated for live cells based on forward and side scatter characteristics were collected for each sample and data were analyzed using CellQuestPro software (Becton Dickinson). For monitoring of DC phenotype, cells were labeled with fluorescent phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) specific for CD1a (Coulter Corporation, Hialeah, FL) and CD11c (Becton Dickinson); and with fluorescein isothiocyanate Targeting Phagocytes by PS-CNT (FITC)-conjugated mouse mAbs specific for CD83 and CD14 (Becton Dickinson) according to the manufacturer’s instructions. Control samples were labeled with isotype-matched antibodies conjugated with the same fluorochrome. Fluorescence was measured with a FACSCalibur flow cytometer (Becton Dickinson) and data were analyzed using CellQuestPro. Fluorescent microscopy Cells were seeded on cover slides 18 h before treatment, 20 mg/ ml of various functionalized SWCNT were added and incubated in DMEM with no phenol red at 37uC for 2 h. At the end of incubation cells were gently washed 3 times with PBS, fixed by 2.5% paraformaldehyde at RT for 5 min and examined under a Nikon ECLIPSE TE 200 fluorescence microscope (Tokyo, Japan) equipped with a digital Hamamatsu charge-coupled device camera (C4742–95-12NBR) and analyzed using the MetaImaging SeriesTM software version 4.6 (Universal Imaging Corp., Down- ingtown, PA). Confocal microscopy RAW 264.7 macrophages were seeded on LabTek chamber slides at a density of 56105 cells per well the previous day. The normal DMEM medium was replaced with phenol red free RPMI 1640 medium the next day and incubated with LysoTracker Red- DND99 (50 nM) for 1 h to label lysosomes. Prior to incubation with LysoTracker Red, RAW 264.7 macrophages (56105 cells per well) were incubated with cocktail of endocytosis inhibitors containing a mixture of nystatin (25 mg/ml), genistein (200 mM), chlorpromazine (6 mg/ml) and brefeldin A (10 mg/ml) for 30 min as described [40]. Cells were then washed twice with PBS followed by addition of various functionalized SWCNT (150 mg/106 cells). The cells were incubated with particles for 5 minutes for the purpose of confocal microscopy. Cells were then washed three times with PBS and fixed using 2% paraformaldehyde. Nuclei were stained with Hoechst 33342. Cells were imaged using Olympus Fluoview 1000 confocal microscope. Following incubation for 2 or 24 h with NBD-labeled SWCNT, MDDCs were fixed in 4% formaldehyde for 15 min. Staining of cell membranes was carried out using anti-HLA-DR mAb (Becton Dickinson). A secondary goat anti-mouse mAb labeled with Alexa Fluor 546 (Molecular Probes, Eugene, OR) was used for detection. Slides were mounted with anti-fading Vectashield mounting medium (Vector laboratories, Burlingame, CA). Fluorescent images were acquired on a confocal laser-scanning microscope (TCS SP2; Leica Microsystems, Mannheim, Germany) equipped with one argon and two HeNe lasers. NBD was excited with a 488-nm laser line detecting light in the wavelength region of 560– 700 nm. Alexa 546 was excited by a 543-nm laser line with detection of light in the region of 560–700 nm. Scanning Electron Microscopy Macrophages fixed in cold 2.5% glutaraldehyde were rinsed in PBS, post-fixed in 1% Osmium Tetroxide (Electron Microscopy Sciences, Hatfield, PA) with 0.1% potassium ferricyanide (Fisher Scientific, Pittsburgh, PA) dehydrated through a graded series of ethanol, from 30 to 100%, and then critical point dried in a critical point dryer, Emscope CPD, (EMScope Lab, Ashford, Great Britain). Following critical point drying, the samples were attached to aluminum SEM specimen mounting stubs (Electron Microscopy Sciences) and then sputter coated with a gold palladium alloy (Sputter Coater 108 Auto, Cressington Scientific Instruments, Cranberry Township, PA). Following processing, samples were analyzed using a JEM 6330F microscope (JEOL, Peabody, MA). PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4398