Fitc conjugated anti mouse cd3

Cell surface staining was conducted with the appropriate fluorochrome-conjugated Abs for 30 min at 4°C. The following specific monoclonal antibodies (mAbs) were used: antigen-presenting cells (APC)-cy7-conjugated anti-mouse CD45, FITC-conjugated anti-mouse CD3, Percy5.5-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8, PEcy7-conjugated anti-mouse NK1.1, BV421-conjugated anti-mouse Nkp46, BV421-conjugated anti-mouse major histocompatibility complex II (MHC-II), APC-conjugated anti-mouse F4/80, PE-conjugated anti-mouse CD11c, PE-CF594-conjugated anti-mouse CD11b and APC-conjugated anti-mouse CD27 (all purchased from BD Bioscience). Flow cytometric (FCM) analyses were performed on a CyAn ADP analyzer (Beckman Coulter, Inc., Kraemer Boulevard Brea, CA, USA), with data analyzed using FlowJo Version 6.1 software (TreeStar, Asland, OR, USA).

Single-cell suspensions were obtained from spleen of mice on indicated days postinfection. For intracellular cytokine staining, cells were stimulated for 5–6 h with PMA (50 ng/mL) and ionomycin (500 ng/mL) in the presence of brefeldin A (10 μg/ml). Cells were harvested, washed, and stained with surface molecule antibodies in the presence of FcR-Block (BD Bioscience). After the wash, cells were then fixed using CytoFix/CytoPerm buffer (BD Bioscience) and stained with antibodies against intracellular cytokines or isotype control on ice for 30 min. The following Abs were used for staining: FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse IL-17A, PE-Cy5.5-conjugated anti-mouse IFN-γ, and APC-conjugated anti-mouse CD4 (BD Bioscience). Treg cell staining was performed using a Treg staining kit according to the manufacturer’s instructions (eBioscience). Data were acquired on a FACS Calibur (BD Bioscience) and analyzed using Flowjo software (TreeStar).

To characterise the leukocyte cell population, spleens were mechanically disrupted, to release single cell suspensions, then filtered through a 40 μm cell strainer (Sigma-Aldrich, Castle Hill, NSW, Australia). Red blood cells were lysed with ammonium-chloride-potassium (ACK) lysing buffer for 10 minute (min) and further filtered using a 40 μm cell strainer to remove cellular debris. Prior to antibody staining, the splenocytes were first incubated with anti-mouse CD16 / CD32 FC block (BD Pharmingen, clone 2.4G2; 1:100) for another 10 min to prevent non-specific background staining via Fc receptors. Then, splenocytes were immediately incubated with FITC-conjugated anti-mouse CD3 (BD Pharmingen, clone 145-2C11; 1:100) and PE-Cy7-conjugated anti-mouse CD45 (eBioscience, clone 30-F11; 1:100) antibodies diluted in FACS buffer (PBS with 2% foetal bovine serum (FBS, Invitrogen, Scoresby, VIC, Australia) for 45 min at 4°C. To terminate the labelling process, cells were washed in FACS buffer and re-suspended in propidium iodide (PI) 20 μg/mL (Sigma-Aldrich, Castle Hill, NSW, Australia) containing FACS buffer immediately before data acquisition using the CyanADP (Beckman Coulter, Lane Cove West, NSW, Australia). Dead cells and debris were excluded from the analysis. All analyses were done using the Kaluza Flow Analysis Software (Beckman Coulter, Lane Cove West, NSW, Australia).