Cd3 cd19

K+-EDTA anti-coagulated whole blood was incubated for 20 min at room temperature with fluorescently labeled antibody pairs (CD45/CD14, CD3/CD19, CD3/CD4, CD3/CD8, CD3/HLA-DR, CD3/CD25, CD57/CD8, CD3/CD16 + CD56, CD8/CD11b, CD19/CD5, and isotype matched controls IgG1 FITC/IgG2a PE, all from Becton Dickinson, Heidelberg, Germany). Erythrocytes were lysed with BD FACS lysing solution according to the manufacturer’s instructions and leukocytes analyzed by flow cytometry on a FACSCalibur using BD Simulset software for data acquisition and analysis. (Becton Dickinson, Heidelberg, Germany). Total leukocyte content was determined by automatic cell counting using a CASY TT (Schärfe System, Reutlingen, Germany).

To characterize the children (at 20 months of age) and their mothers in regard to the profile of T (helper and cytotoxic) and B lymphocytes, we used total leukocytes obtained from 100 µL of hemolyzed peripheral blood with an RBC Lysis solution (QIAGEN, Hilden, Germany). After this step, the cells were incubated with specific antibodies conjugated to fluorochromes at 4 °C for 30 min. After labeling, the samples were washed with a phosphate-buffered solution and taken for analysis by flow cytometry.
T and B lymphocyte populations were characterized by CD3/CD4/CD8 and CD3/CD19 (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) panels of specific antibodies, respectively. Data acquisition was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA), and data analysis was performed using FlowJo software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The percentage of cells expressing the markers and their expression levels, indirectly determined by the mean fluorescence intensity (MFI), represent the main results used to analyze ZIKV infection repercussions in T and B cell populations.