1% sodium azide and 0.05 mM EDTA and resuspended in the same buffer to a density of 5 × 106 cells/ml. The following anti-mouse monoclonal antibodies directed against surface antigens were used: TcR1-FITC (clone GL3) from AbD Serotec and CD19-PE-Cy5.5 (clone 6D5), CD3-APC (clone 145-2C11),
CD45-FITC (clone 30-F11), CD16/32-PE (clone 93) and CD14-FITC (clone Sa2-8) from eBioscience. Before the flow cytometry, the isolated lymphocytes were incubated with the appropriate antibodies for 30 min, washed twice in PBS and analyzed by FACSCalibur™ (BD Biosciences) equipped with a 488 nm argon-ion laser and a 633 nm diode laser. At least 105 cells were analyzed and data analyses of gated lymphocytes positive for CD45 were performed using CELLQuest™ Pro software (BD Biosciences). γδ T-lymphocytes
were identified in a single TcR-specific staining. CD19-positive B-lymphocytes and CD3-positive T-lymphocytes, and CD4 and CD8 Th- and Tc-lymphocytes, were each characterized click here by separate two-colour analysis. Finally, the CD14 and CD16 positive cells out of CD3 and CD19 double negative were quantified using a four-colour analysis. Real time PCR Total RNA was extracted from caecal wall samples using the RNeasy Lipid Tissue Kit (Qiagen). Resulting RNA was eluted with 50 μl RNase-free water and used immediately in reverse transcription using M-MLV reverse transcriptase (Invitrogen) and oligo-T primers. The resulting cDNA was purified by the QiaPrep PCR Purification kit (Qiagen) and used as a template for quantitative PCR. mRNA expression rates of TNFα, Belnacasan supplier IL-12p40, IL-18, IFNγ and iNOS were determined using the QuantiTect™ SYBR® Green RT-PCR Kit (Qiagen) with β-actin mRNA as a reference. Primers used for the RT-PCR are listed in Table 4. The threshold cycle values (Ct) of gene of interest were first normalised to the Ct value of actin
reference mRNA (ΔCt) and the normalised mRNA levels were calculated as 2(-ΔCt). The normalised mRNA levels of a particular cytokine were then used for t-test comparisons between the infected and non-infected animals and are also given in figures as “”actin”" units. Table 4 List of primers used for the quantification of gene expression by real time RT PCR. primer sequence 5′-3′ length (bp) Reference TNFαFor CATCTTCTCAAAATTCGAGTGACAA 175 [34] TNFαRev TGGGAGTAGACAAGGTACAACCC IL-12p40For GGAAGCACGGCAGCAGAATA 180 [34] IL-12p40Rev oxyclozanide 10058-F4 research buy AACTTGAGGGAGAAGTAGGAATGG IL-18For CAGGCCTGACATCTTCTGCAA 105 [34] IL-18Rev TCTGACATGGCAGCCATTGT IFNγFor AACAGCAAGGCGAAAAAGGA 92 this study IFNγRev GTGGACCACTCGGATGAGC iNOSFor CAGCTGGGCTGTACAAACCTT 95 [34] iNOSRev CATTGGAAGTGAAGCGTTTCG β-actinFor CTTTGCAGCTCCTTCGTTG 150 this study β-actinRev ACGATGGAGGGGAATACAGC Statistical analysis Data were evaluated by parametric two-sample, equal variance, t-test and non-parametric Mann-Whitney test comparing the experimental groups either to the non-infected control mice or to the mice infected with the wild type S. Enteritidis.
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