Fitc conjugated anti mouse cd19

Cells were washed twice in phosphate buffered saline (PBS) and resuspended at 1 × 10⁶ cells/ml in FACS buffer (PBS/0.1% NaN₃/1% FBS). Cells were blocked with rat anti-mouse CD16/CD32 antibody (BD Biosciences) at 4°C for 5 min and then stained with fluorescein-conjugated anti-mouse SR-AI, PE-conjugated anti-mouse LOX1 (R&D Systems, Minneapolis, MN, USA), PE-conjugated anti-mouse CD36, FITC-conjugated CD11b, PE-conjugated anti-CD11b, PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD19, and FITC-conjugated anti-mouse IA/IE (BD Biosciences) (all antibodies were diluted 1:100) for 30 min on ice in the dark. Matched isotype antibodies were used to show nonspecific binding. The cells were washed and resuspended in FACS buffer. A total of 10,000 events were acquired on a Navios flow cytometer (Beckman Coulter, La Brea, CA, USA), and the data were processed using Kaluza software (Beckman Coulter).

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).