Pecy7 conjugated anti mouse nk1

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).

To analyze PTX3 intracellular expression, transfected HEK 293T cells were fixed with 2% PFA and permeabilized with 0.1% Saponin (Sigma Aldrich) in PBS. Indirect intracellular staining was performed with rat anti-PTX3 (MNB4, Abcam) primary antibody, followed by AF488-conjugated anti-rat (Life Technologies) secondary antibody. To identify the various cell populations present in splenocytes, peritoneal lavage and quadriceps harvested from mice, cells were first incubated with anti-mouse CD16 / CD32 (FC block, BD Pharmingen) and stained with the following antibodies: APC-conjugated anti-mouse GR1, PE-conjugated anti-mouse F4/80, FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse Ly6c, APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD19, PE-conjugated anti-mouse CD45, or PE-Cy7-conjugated anti-mouse NK1.1 (BD Pharmingen). For detection of alphavirus antigens, indirect intracellular staining was performed using mouse monoclonal anti-alphavirus (3581, Santa Cruz) primary antibody, followed by AF488-conjugated anti-mouse (Life Technologies) secondary antibody. Data acquisition was performed using CyanADP (Beckman Coulter), and analysis was done by Kaluza Flow Analysis Software (Beckman Coulter).