conditioned units (25uC, 50% relative humidity) and kept on a 12 h light/dark cycle. Water and mouse chow were provided ad libidum. All the experimental protocols were approved by JNU Institutional Animal Ethics Committee (IAEC Project Code: 5/ 2010) and performed accordingly. Cells and Reagents For in vitro studies, erythrocytes derived from mouse blood were suspended in RPMI complete medium (RPMI-CM) containing glutamine (2 mM), HEPES buffer pH 7.2 (25 mM), gentamycin (20 mg/ml) obtained from Sigma-Aldrich (India) and fetal bovine serum, obtained from Hyclone (South Logan, UT). ANS (8-Anilino-1-naphthalenesulfonic acid) was obtained from Sigma-Aldrich (India). Alexa fluor 488/633 hydrazide was obtained from Molecular Probes (Carlsbad, CA). Biotin-X-NHS Ester (BXN) was from Calbiochem (La Jolla, CA) and Streptavidin Allophycocyanin (SAv-APC) and Annexin-V PE were obtained from BD biosciences (San Diego, CA). Single-wall carbon nanotubes (SWCNT’s) were purchased from Sigma (Catalogue #: 636797, amorphous carbon ,3%). Similar results were obtained for SWCNTs preparations procured from [Carbon Nanotubes Inc. Houston, TX, USA (purified HIPCO SWCNTs ,15% ash)] and their derivatives. Acid functionalization of particles Acid functionalized SWCNTs (AF-SWCNTs) were produced by suspending 20 mg of powder SWCNTs in 20 ml of 1:1 concentrated HNO3:H2SO4 in 100 ml high-pressure vessels in a microwave digester as described previously [5]. Briefly micro- wave power was applied at 50% of 900 watt total and the pressure was controlled at 2062 psi for 3 min resulting in an internal temperature of 138–150uC. Suspensions were cooled, diluted five-fold with H2O and dialyzed four times against 5 liter water over a two day period. Dialyzed suspensions were freeze dried, weighed and resuspended in 5 ml water. Particle size distribution and surface charge on AF-SWCNTs were as reported before [5]. The particle preparations were sonicated for 1 min in ice using a sonicator (Branson sonifier, VWR Scientific) prior to use. Attachment of fluorescent probes to AF-SWCNT For covalent attachment of fluorescent probes to the nanotube surface, -COOH groups on AF-SWCNTs were exploited. AF- SWCNTs were suspended in water and treated with 1-ethyl 3-(3- dimethyl aminopropyl) carbodiimide (EDAC) and N-hydroxy succinimide (NHS) in order to get a succinimidyl intermediate. The mixture was continually shaken for 2 hrs and dialysed thereafter to remove excess NHS, EDAC and urea by-product. AF-SWCNTs thus activated were incubated with Alexa Fluor 488/633 hydrazide (Molecular Probes, Carlsbad, Ca) in dark with continuous mixing, followed by dialysis to remove free dye [7]. Attachment of fluorescence tag to AF-SWCNTs was confirmed flow cytometry. Confocal Microscopy of erythrocytes Attachment or internalisation of AF-SWCNTs was confirmed by incubating erythrocytes with alexa fluor 488/633 tagged AF- SWCNTs, as described previously [7]. Erythrocytes were incubated in RPMI+1% Fetal Bovine Serum with fluorescenated AF-SWCNTs for 1 h and unbound/loosely bound particles were removed by three extensive washes with PBS, after that suspensions were analysed by Confocal Laser Scanning Micro- scope (Olympus FluoviewTM - FV1000). Effect of Carbon Nanotubes on Erythrocytes Measurement of membrane damage Hydrophobicity of the erythrocytes was determined by staining with 8-anilino-1-naphthalenesulfonic acid (ANS) as described before [8]. Erythrocytes suspended in PBS were incubated with ANS (4 mM) at 37uC for 20 min. The binding of ANS to hydrophobic sites on erythrocyte membrane was measured (Exmax = 365 nm) using a Fluorimeter (Spectramax M2e). Fluo- rescence spectra were scanned from 400 to 600 nm. The excitation and emission band passes were 5.0 nm in width. Particle Exposure Erythrocytes were suspended in RPMI 1640 supplemented with 10% Fetal bovine serum and incubated with control and AF- SWCNT’s at different concentrations over rotisserie (17 RPM) at 37uC for different time intervals. For in vivo studies, mice were given 100 mg AF-SWCNTs or saline control through intravenous, intraperitoneal, gavage and intratracheal routes and blood samples were taken at several time points thereafter. Blood erythrocyte count was determined by automated hematology analyser (Melet Schloesing Laboratories MS4e, Osny, France). In vivo biotin labelling In vivo biotinylation of circulating erythrocytes was done as described previously [9], [10], [11], [12]. All blood erythrocytes were biotinylated by three daily intravenous injections of 1 mg of biotin-X-NHS Ester (BXN) dissolved in 20 ml of dimethylforma- mide (DMF) and 250 ml of phosphate buffered saline (PBS). Thirty days later, a second lower dose of B-X-NHS (0.6 mg) labelled all erythrocytes generated over thirty days with low intensity biotin. Blood erythrocytes derived from these mice 10 days after the second biotinylation step were used to enumerate old (biotinhigh population, age.40 days), intermediate (biotinlow, age 10 to 40 days), and young (biotinnegative, age ,10 days) populations of erythrocytes. Biotin high, low and negative erythrocyte populations could be identified and enumerated by staining with streptavidin-APC followed by flow cytometry as described before [9]. Flow cytometry To enumerate erythrocytes with high, low or no biotinylation, 16106 erythrocytes were stained with streptavidin allophycocya- nin (APC) as described before [9]. For PS externalisation studies, erythrocytes were stained with annexin V-PE, by the procedure recommended by the manufacturer (BD Biosciences). Briefly, erythrocytes were suspended in annexin-V binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and annexin-V PE for 20 min at 25uC in dark. Stained erythrocytes were immediately analysed on FACS Calibre flow cytometer (Becton Dickinson, San Jose, CA, USA) using Cell Quest software for acquisition and analysis. A minimum of 10,000 events were recorded for each sample. Statistical analysis Each experiment was repeated at least three times. Statistical analysis by two-way ANOVA and student’s t-test were done using Sigma plot and Sigma stat software. Data are presented as means 6 SEM. Results Effect of control and acid functionalized SWCNTs on the recovery of murine erythrocytes in culture The effect of control and acid-functionalized SWCNTs was examined on murine erythrocytes. For this purpose, blood PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22032