2.6. Assessment of neutrophils activation markers by flow cytometry analysis

CD62L and CD66b are known to be, respectively, downregulated and upregulated on PMNs upon stimulation/activation.18 PMNs uninfected and infected with L. (V.) braziliensis, or stimulated with ionomycin (500 ng/mL) plus 10 ng/mL of phorbol‐myristate‐acetate (PMA), were cultured for 90 minutes at 37°C and 5% CO₂. Cells were stained with PE‐conjugated MAb α‐CD62L, APC‐conjugated MAb α‐CD66b and PE‐Cy7‐conjugated MAb α‐CD15 (BD Pharmigen). PMNs were stained with MAbs in diluent buffer (20 minutes at 4°C) washed two times with PBS 1X and then fixed with 2% formaldehyde. About 20 000 events were gated, acquired by FACS Calibur (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and analysed by Flow Jo Software (Becton Dickinson). The frequency of double positive cells CD62L⁺/CD15⁺ and CD66b⁺/CD15⁺, and the mean of fluorescence intensity (MFI) were determined followed FACS strategy shown in Figure 3A.