Anti tlr2 pe

Cells were stained with anti-CD3-PerCP Cy5.5 (eBioscience), anti-CD8-APC (BD Bioscience, San Jose, CA), anti-TLR2-PE (eBioscience), anti-TLR7-FITC (eBioscience), anti-lymphocyte activation gene-3 (LAG-3)-PerCP (BD Bioscience), and anti-CD279 (programmed death-1, PD-1)-FITC (BD Bioscience) following manufacturer’s instructions. Acquisitions were performed using CellQuest Pro Software by FACS Calibur (BD Bioscience), and data were analyzed using FlowJo V10 (TreeStar, Ashland, OR).

The samples were submitted to the treatments described above and analyzed for the expression of the cell surface markers CD11b, CD14, C5aR1, C3aR, TLR2 and TLR4 on the surface of leukocytes labeled with specific antibodies for the monocyte (CD33) and granulocyte (CD66b) populations. After the blood treatment, red blood cells were lysed with BD FACS Lysing Solution buffer (BD Biosciences, California, USA). Subsequently, the cells were centrifuged at 405 g at 4°C for 10 minutes, resuspended and marked with monoclonal antibodies from BD Biosciences (California, USA) or eBioscience (California, USA), which were diluted in a 1:5 ratio with anti-CD11b PE (VIM12 clone), anti-CD14 FITC (clones 61D3 and TüK4) and anti-CD33 APC; in a 1:10 ratio with anti-C5aR FITC (clone 8D6), anti-C3aR PE (clone 17), anti-TLR2 PE (clone TL2.1) and anti-TLR4 PE (clone HTA125); and in a 1:20 ratio with CD3 APC-Cy7, CD19 PE-Cy7 and CD66b Alexa647. Monoclonal mouse IgG1k PE and IgG2ak FITC mice were also used as isotypic controls. After 30 minutes of incubation, 275 μL of FACS buffer was added, and the cells were analyzed in a FACSCanto II flow cytometer (BD Biosciences, California, USA) using SOFTWARE BD FACSDiVa, version 4.1 (BD Bioscience, California, USA). The results were expressed as median fluorescence intensity (MFI), determined from the acquisition of 20,000 events.