temperature using 2 mM Sulfo-NHS-LC-Biotin (Pierce Biotech- nology). Cells were washed three times with PBS containing 100 nM glycine and once in PBS. Labelled cells were lysed in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitors and 1% Brij 97 (Sigma-Aldrich). Lysates were cleared by centrifugation for 15 min at 12,000 g and sequentially incubated at 4uC in the presence of Protein-G Sepharose (Gamma bind Plus Sepharose, Pharmacia), Protein G Sepharose-bound normal mouse IgG (Jackson Immunoresearch) and Protein G Sepharose-bound anti- cdTCR. Immunoprecipitated samples were washed three times with lysis buffer then boiled in reducing sample buffer (30 mM Tris, pH 6.8, 5% glycerol, 2% SDS, 2.5% b-mercaptoethanol, 0.1% bromophenol blue). Material was run on 14% polyacryl- amide gels. Material corresponding to the total and to 1/10th of the material was loaded for eosinophils and cdT cells respectively. Following separation, proteins were transferred to PVDF mem- brane (Biorad). Blots were developed using ABC-HRP (Vector Laboratories) and the enhanced chemiluminescence (ECL) plus Western blot detection system (Amersham). PCR analysis Total RNA was isolated using the RNeasy mini kit (Qiagen) from 107 purified cells and reverse transcription was performed using SuperScriptTMRT (MMLV 200 U/ml) (Invitrogen). cDNA was amplified using primers (Proligo) (20 pmoles/ml) listed in Table S2. PCR were run for 40 cycles (1 min at 95uC, 1 min annealing and 1 min at 72uC) using Taq polymerase (Bioprobe). Real time PCR Total RNA was prepared from eosinophils by guanidium/CsCl centrifugation method. Briefly, purified eosinophils were pelleted by centrifugation and then lysed in 4 M guanidium isothiocyanate, 1 mM EDTA, 25 mM sodium acetate, 4.9% b-mercaptoethanol, 68 mM N-lauryl sarcosine. Lysate was drawn through a 20G needle. RNA was obtained by ultracentrifugation (28000 rpm, 20 h, and 20uC) through a CsCl gradient. RNA pellet was washed twice and dissolved in water and precipitated overnight at 220uC with ethanol 70% and sodium acetate 0.08 M. After centrifugation (10000 rpm, 45 minutes at 4uC), RNA was washed twice in ethanol 70%, resuspended in water and store at 280uC until use. RNA from cd T cells and Colo205 cells was prepared using RNeasy mini-spin columns (Qiagen, UK) according to the manufacturer’s instructions. All samples were quantified by absorbance measurement at 260 nm on a spectrophotometer (Biorad), and RNA quality was checked by running samples on 1.5% agarose gel in RNA loading buffer (Sigma). Total RNA was first submitted to DNAse I (Invitrogen) treatment (15 min at room temperature), and cDNA was generated using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer instructions. Samples were analyzed by quantitative real-time PCR, performed according to manufacturer’s protocol, using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Primers were designed using the Primer3 Website and are listed on Table S2. Samples were run in triplicate in a reaction volume of 25 ml. Amplification was carried out for 40 cycles, with denaturation at 95uC for 10 min during the first cycle and subsequently for 15 seconds, annealing and extension for 1 minute at 60uC. A dissociation temperature gradient was included at the end of the run. Gene expression was normalized according to GAPDH expression. Relative gene expression was calculated with the 22DDCT method [65]. Eosinophil cdTCR Expression TCR repertoire analysis Qualitative analysis of CDR3 length for Vc9-Jc1/2, Vc9-JcP and Vd2-Cd families was performed in triplicate on eosinophils and in cd T cells from the same donor (TcLand, Nantes, France) [66]. Briefly, cDNA was amplified by PCR using a Cc or Cd reverse primer and Vc9 or Vd2-specific forward primers, respectively. The amplifications were performed in a 384-Well GeneAmpH PCR System 9700 (Applied Biosystems, Foster City, CA). Briefly, each amplification product was used for an elongation reaction using a dye-labeled Jc specific-labelled primer for the rearrangement analysis and Cd labeled primer for the d chain analysis, then heat denatured, loaded at least in duplicate onto a sequencing analyzer (48-capillary 3730 DNA Analyzer - Applied Biosystems). Then, GeneMapper software (Applied Biosystems) was used to display the distribution profiles of CDR3 lengths, in amino acids, of the amplified and elongated products. cdTCR junctional sequences analysis Vd1-Jd3 and Vd2-Cd amplification products (400 bp) were cloned into a TA vector (pCR-TOPO, Invitrogen) using standard protocols. For each rearrangement, sequencing was performed on 5 randomly picked clones. TCR sequences were obtained from the IMGT website (imgt.cines.fr). Sequence alignments were per- formed using DNAstar software. GeneBank sequence accession numbers are: FJ890312 and FJ890313 respectively. In situ hybridisation PCR products, amplified from purified cdTCR lymphocyte RNA, and corresponding to VcI-JcP, Vd2-Cd and Vd1-Jd4 rearrangements were cloned into the pCRII-TOPO vector (Invitro- gen). Clones were isolated and sequenced on both strands. Clones corresponding to the published sequence were used to generate hybridization probes. Sense and anti-sense probes were synthesised from linearised plasmids using 350 mM digoxigenin-UTP (Roche) and SP6 or T7 polymerases as described [67]. In situ hybridisation was performed using a Discovery automat and corresponding kits (Ventana Medical Systems). Slides were incubated in EZprep buffer before processing with a standard RiboMap Kit. Slides were pre- treated 30 min with CC2 buffer, then 20 min with protease 3, followed by 6 h hybridization with sense or anti-sense probes (100 ng/slide). Slides were then washed twice 10 min in 26 SSC at 60uC, twice 10 min in 0.16SSC at 60uC. Slides were incubated for 30 min with a mouse anti-digoxigenin antibody (Jackson Immu- noResearch) and then for 30 min with a rabbit anti-mouse antibody before treatment with UltraMap kit. Labelling was detected after a 150 min (Vd2-Cd), 110 min (VcI-JcP) or 90 min (Vd1-Jd4) incubation in the presence of NBT/BCIP substrate. Slides were counterstained for 1 h with Nuclear Fast Red and mounted in permanent mounting medium (Vector). ROS production and EPO release ROS production and EPO release were measured as described [61]. Wells were coated with 10 mg/ml anti-mouse IgG F(ab’)2 (Sigma) for 2 h at 37uC, followed by incubation with 10 mg/ml anti-CD3, anti-cdTCR, anti-Vd1or isotype control mouse IgG1 for 2 h at 37uC. Eosinophils (26106/ml) were added in the presence of luminol (8.3 mg/ml). Alternatively, eosinophils were stimulated with 50–200 nM BrHPP, 5–50 mM sec-butylamine (SBA) (Sigma) or IgA/anti-IgA immune complexes (incubation with 7.5 mg/ml secretory IgA for 1 hr then by addition of 10 mg/ ml anti-IgA). Chemiluminescence was measured at 37uC for 5 sec/timepoint with a luminometer (Victor2TM Wallac). For EPO release, supernatants (50 ml) were transferred into a microtiterplate PLoS ONE | www.plosone.org 14 June 2009 | Volume 4 | Issue 6 | e5926