4.7. Flow Cytometry

To determine the expression level of the fMLF receptors FPR1 and FPR2, isolated PMNs were diluted to a concentration of 3 × 10⁶ cells/mL in staining buffer (D-PBS + 2% fetal calf serum) and added to a 96-well plate. After addition of anti-FPR1, anti-FPR2 or PE-labeled anti-CD16 antibody, the cells were incubated for 30 min on ice in the dark. Subsequently, cells were washed three times with staining buffer, and the cells incubated with unlabeled antibodies were labeled with PE-labeled goat anti-mouse antibody and again incubated for 30 min on ice in the dark. The cells were washed 3 times with staining buffer and finally fixed with fixation buffer (staining buffer + 0.4% formaldehyde). FPR1 and FPR2 expression was measured with an FACSCalibur flow cytometer (BD Biosciences). The results were analyzed by CellQuest software (BD Biosciences).