Igg1 fitc

The following details concerning the antibodies (Abs) and reagents were used in this study: mouse anti-chicken [MHC-class II⁺ (clone-2G11), CD86⁺ (Clone-GL1), CD1.1⁺ (Clone-CB3), CD11c⁺ (Clone-N418)] with appropriate isotype IgG1-PE, IgG2a-PE/Cy7, IgG1-FITC, IgG-APC, respectively, all were from Southern Biotech (Birmingham, AL). Hiscript II Q RT SuperMix for QRT-PCR (⁺gDNA Wiper) (L/N: 7E312C9) and ChamQ SYBR QRT-PCR Master Mix are from Vanzyme. TRIzol (L/N: A304-1) from TaKaRa Biotechnology (Dalian) Co. Ltd., Dalian City, Liaoning, China. The enhanced horseradish peroxidase (HRP)-DAB chromogenic Kit (L/N: S7418) was from TIANGEN Co. Ltd. (Tiangen, China). Freund's adjuvant Complete and Freund's adjuvant Incomplete were from (Sigma Aldrich, Merck KGaA, Darmstadt, Germany). Lymphocyte separating medium (L/N: LTS1077) was from TBD-science, Tianjin, China. Gibco RPMI Medium-1640 (L/N: 2037577) and Chicken Serum was from Life Technologies, San Francisco, CA. Recombinant chicken-granulocyte macrophage colony-stimulating factor (GM-CSF) was from Abcam, Cambridge, UK, and recombinant chicken interleukin 4 (IL-4) was from Kingfisher, London, UK.

The cellular mucosal immune system status of the broiler's intestinal tract, intraepithelial lymphocytes (CD3+ T lymphocytes, CD3+CD8+ cells, CD3+CD4+ cells, and CD3+CD4+CD8 cells) were measured. At post mortem, intraepithelial T lymphocytes were estimated from isolated jejunum of 40 birds (1 bird per cage) at 42 d of age according to the method defined by Röhe (2014) . The intestinal cell suspensions were stained with either a cocktail of T lymphocyte CD marker antibodies (CD3-AF647, CD4-FITC and CD8-PE; Southern Biotech, Birmingham, AL, USA) or a cocktail of isotype control antibodies (IgG1-PE, IgG1-FITC, and IgG1-AF647; Southern Biotech, Birmingham, AL, USA) for 30 min on ice in the dark according to the manufacturer's instructions.
Flow cytometric data were acquired on an Amnis FlowSight imaging flow cytometer (Millipore, Burlington, Massachusetts, USA). CD4-FITC and CD8-PE were detected using the 488 nm laser, and the CD3-AF647 was detected using the 633 nm laser. Signals from the isotype antibody cocktail were subtracted from the T lymphocyte CD antibody fluorescence. From the total cell population that was acquired, the CD3+ intact cell population was gated. Sub-gates were applied to the intact CD3+ cells population to determine the proportion of cytotoxic T lymphocytes (CD3+CD8+), T helper lymphocyte (CD3+CD4+) and double-stained T lymphocytes (CD4+CD8+).