molecular mechanisms of differentiation, diversity and function of T-cell expressed cdTCR. Materials and Methods Ethics statement Peripheral venous blood was obtained from healthy donors or eosinophilic patients after written informed consent. Research protocol was approved by the Comite´ Consultatif de Protection des Personnes dans la Recherche Biome´dicale de Lille (France). Cell purification and culture Eosinophils were isolated as described [61] on Percoll followed by negative selection using anti-CD16-coated microbeads. Purity determined after cytospin and RAL555 staining was above 98% and 100% for RNA analyses. Purified eosinophils were cultured overnight at 37uC in complete RPMI without phenol red. Neutrophils were obtained by positive selection with anti-CD16 coated microbeads. Purity was 99%. For RNA extraction, abTCR lymphocytes from PBMC from normal donors were sorted after staining with anti-abTCR-PE using FacsAriaTM cell sorter and DivaTM software (BD). Purity was 99%. cdTCR lymphocytes were generated from PBMC (0.56106/ml) by culture in complete RPMI 1640 medium with 200 nM bromohydrin pyrophosphate BrHPP (Innate Pharma, Marseille) and 20 ng/ml rhIL-2 for 7 days. At that time, 50–70% cells were c9d2TCR+ cells. For RT- PCR and in situ hybridization, cdTCR+ cells were further purified using anti-cdTCR microbeads (Miltenyi). Cord blood was obtained from the maternity unit of Lille University Hospital. Mononuclear cells were isolated by Ficoll- Hypaque density centrifugation. CD34+ cells were purified using CD34+ isolation kit (Miltenyi). Purified CD34+ cells were cultured in complete medium supplemented with 2.561025 M b-mercap- toethanol (b-ME) at 0.56106 cells/ml. Eosinophil differentiation was induced upon addition of 50 ng/ml SCF, 1 ng/ml each GM- CSF, IL-3 and IL-5 (Peprotech). Colo-205 colon carcinoma cell line was obtained from ATCC. Cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 10 mM L-Glutamine and 10 mg/ml Gentamycin. Flow cytometry Antibodies and isotype controls used for flow cytometry (origin, clone, working dilution or concentration) are listed in Table S1. Anti-EPO (AHE) and anti-MBP2 [62] (a kind gift from Dr. D Plager, Mayo Clinic, Rochester) were biotinylated using biotin- sulfosuccinimidyl ester (Molecular Probes) according to manufac- turer’s instructions and used at a 1:100 dilution. Samples were analysed on a FACSCaliburTM using CellQuestTM software (BD). Multiple staining experiments are represented as color density plots [63] with linear scales used for both forward (FSC) and side scatter (SSC) and logarithmic scales used for fluorescence parameters (Log density: 50%). Intracellular staining was performed after fixation of purified eosinophils, neutrophils or PBMC (26105) with 2% paraformal- dehyde and permeabilization with 0.01% saponin in PBS. After blocking of non-specific binding with 5 ml mouse serum for 10 min, cells were incubated with anti-CD3-FITC, anti-abTCR- FITC, anti-cdTCR-FITC or anti-CD8-FITC or corresponding isotype controls for 30 min in the presence of saponin. Following incubation for 2 h with or without TUBag-1 [36] (40 nM) in RMPI 1640 or after overnight culture, purified blood eosinophils were gated prior to the analysis, on the basis of their FSC and SSC characteristics in order to exclude dead cells (Figure 1B). Within PBMC fraction, monocytes and lymphocytes Eosinophil cdTCR Expression were gated on the basis of their FSC and SSC characteristics prior to the analysis. Quadrants were set on samples after double staining with FITC- or APC- and PE- or biotin-conjugated control isotype antibodies. At least 104 events were acquired per sample. For double and triple staining, cultured peripheral eosinophils were preincubated with 5% human serum albumin (HSA) for 30 min at 4uC and labelled for membrane receptor with the relevant fluorochrome-conjugated antibodies. After washing, cells were fixed 10 min with 2% paraformaldehyde at 4uC and permeabilized for 10 min at room temperature (RT) with 0.01% saponin in PBS-1% BSA. Non-specific binding was blocked with 5 ml mouse serum for 10 min. Purified peripheral blood eosinophils were further identified by incubation with biotinylated anti-EPO or anti-MBP or isotype control for 30 min and streptavidin-PE (Molecular Probes) (1:200) for 20 min in the presence of saponin. Neutrophils were identified by intracellular staining with anti-MPO-PE following an identical procedure. Monocytes and T lymphocytes were identified by staining with anti-CD14-PE, anti-abTCR or anti-cdTCR respectively. Immunofluorescence and confocal microscopy Purified eosinophils were cytocentrifuged, fixed in 4% parafor- maldehyde, washed in 0.05 M PBS. Fixation was stopped with 0.1 M glycine for 4 min and 50 mM NH4Cl pH 7.4 for 15 min. After each incubation step, cytospins were washed for 10 min in PBS. Cells were incubated with PBS-3% BSA-5% HSA for 30 min and, after one PBS wash, with 10% decomplemented donkey serum (Jackson ImmunoResearch). Cytospins were then incubated overnight at 4uC in a humid chamber with goat antibodies specific for CD3e, CD3f, CD3c, CD3d, aTCR, bTCR, cTCR, dTCR (Santa Cruz) or with goat IgG (Jackson ImmunoResearch) in PBS-BSA-HSA (40 mg/ml). After washing in PBS-0.1% BSA, cells were incubated with biotinylated donkey anti-goat IgG (Jackson ImmunoResearch) (1:200) in PBS-BSA- HSA for 2 hours, followed by streptavidin-Texas Red (Molecular Probes) (1:500) in PBS-3% BSA for 1 hour. Final washes were with PBS-0.1% BSA- 0.02% Tween 20 for 2610 min and with PBS for 10 min. For immunofluorescence, DNA was stained with Hoechst 33342 (Invitrogen) (1:1000), immediately prior to cytospin mounting in Fluoromount G (Southern Biotechnologies). For conventional immunofluorescence a Leica DM-RB micro- scope equipped for epifluorescence with a 100 W mercury lamp was used. Filter and dichroic mirror sets were TX2 for Texas Red and A for Hoechst 33342. Images were acquired with a 10061.32 oil immersion objective, a Photometrics Cool SNAPTM camera using RSImageTM software (Roper Scientific). Samples were analysed by confocal microscopy using a DM-IRE2 inverted microscope with SP2-AOBS scan-head (Leica) at the Imaging Facility of Institut Pasteur de Lille. Excitation was performed at 543 nm for Texas Red. Fluorescence emission wavelengths pass bands were selected between 581 and 621 nm according to the emission spectral analysis. Excitation power was between 100 and 400 mW. Acquisitions were performed using a 10061.4 oil immersion objective. 3D pre-treatment, analysis and edition were performed with Edit3D (free software, Y. Usson, GDR2588, CNRS, France). Analysis and editing was performed on Photo- shop (Adobe). Surface immunoprecipitation Surface labelling and co-immunoprecipitation was performed as previously described for cd T cells [64]. Purified human eosinophils or differenciated cdT cells were washed and then resuspended in PBS (pH 8.0) at 3.107 cells/ml at room temper- ature. Surface biotinylation was performed for 30 min at room PLoS ONE | www.plosone.org 13 June 2009 | Volume 4 | Issue 6 | e5926