In this scenario, hNPs continue to remain in the vitreous cavity

e; the obtained cell suspension was passed through a cell strainer to eliminate clumps and small fragments. Post-centrifugation supernatant was discard, and cell pellet resuspended in red blood cell lysing buffer. After centrifugation, supernatant was discard and cell pellet resuspended in staining media. A cell count and viability analysis were then performed. Flow cytometry studies were performed using a FC500 or a CyAn flow cytometer. Data were analysed using CXP software or FlowJo software. Antibodies for flow cytometry experiments were obtained from Caltag, BD Pharmingen,, Beckman Coulter and Miltenyi Biotec. CD3+-, CD4+- or nave CD4+CD62L+ T cells were enriched out of thymus or pooled lymph nodes and/or spleen cells suspensions as indicated, with negative selection magnetic cell sorting kit: pan T cell isolation kit mouse, CD4+ T cell isolation kit II mouse, CD4+CD62L+ T cell isolation kit II, respectively. Cell PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729642 cycle and D-α-Tocopherol polyethylene glycol 1000 succinate proliferation analysis Cell cycle analysis of LN T cells was performed using Coulter Reagents Kit on the basis of DNA staining with propidium iodide as described. At least 30000 events were analyzed at low speed and collected on list mode files. The percentage of T cells in the different phases of the cell cycle was determined. T lymphocyte proliferation capacity was analyzed after 24, 48 and 72H of culture of T cells with plate bound anti-CD3- with or without recombinant anti-CD28, with or without recombinant IL-2 by enumeration in a haemocytometer after dilution of T cells in trypan blue, or by cytometric analysis of stimulated LN T cells labeled with the fluorescent dye carboxyfluorescein diacetate succinimidylester . Propidium iodide staining was performed in CFSE labelled cells. In each histogram, the percentage of the dividing cells per cell generation was determined by quantification of CSFE cellular fluorescence halving using a flow cytometer CyAn, and data were analyzed using FlowJo software. Cytokine production and IL4I1 activity analysis The amount of IL2 in 18H culture supernatant of T cells stimulated with ConA, a mitogenic lectin that mimic anti-CD3 stimulation, was quantified by a CTLL2 bioassay, and the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729663 amount of IL-4 and IFN was quantified by Immunoassay.The amount of IFN, IL4, IL10 and IL17A produced in 5-days culture supernatant of plate bound anti-CD3- stimulated nave CD4+CD62L+ T cells with or without recombinant anti-CD28 was quantified by Luminex assay. IL4I activity was quantified as already described by us. Western blot NP-40 total cell lysates of stimulated T cells were resolved on SDS-PAGE and transferred to 0.45m pore size Immobilon-P PVDF membrane. Ab specific for phospho-ERK1/2, ERK1/2, Akt, phospho-Akt, cyclin E, or pRb and actin were used for immunoblotting, and immunoreactive bands were detected using ECF Western Blotting Reagent Pack, and analysed using a Storm Phosphorimager and the Image Quant software. 3 / 18 PEA-15 Rgulates Th Cytokines Expression Immunofluorescence Stimulated purified LN T were spread on Superfrost plus slides at 1×105 cells/slide after washing with PBS containing a cocktail of protease inhibitors: okadaic acid, NaF, sodium orthovanadate, and then fixed and permeabilized by PBS containing 4% formaldehyde for 15 minutes at room temperature. For antiERK1/2 staining, non-specific sites were blocked with goat serum in PBS Triton X-100, incubated overnight at 4C with anti-ERK1 in PBS-Triton X-100 and BSA., and subsequently for 1H at room temperature with Cy3 conj