Anti cd14 pc5

EDTA anticoagulated peripheral blood was used for phenotype analysis by flow cytometry. In detail, 200 μL of whole blood was lysed in 2 mL of VersaLyse at room temperature for 15 min. Then, cells were washed twice with FACS buffer, followed by resuspension in the appropriate antibody preparation and incubated for 20 min at 4°C. For intracellular staining of Ki-67 expression, we used permeabilization and fixation method using eBioscience perm/fix kit (ebioscience cat no. 88-8824-00). The following monoclonal antihuman antibodies were used in appropriate concentrations to stain cells: anti-CD45-Krome orange (Beckman Coulter, cat no. 96416), anti-CD14-PC5.5 (Beckman Coulter, cat no. A70204), anti-CD 19-APC-Alexa fluor 750 (Beckman Coulter, cat no.A94681), anti-CD27-PE (BD Pharmingen, cat no.555441), anti-CXCR3-APC (BD Pharmingen, cat no. 561732), anti-CXCR4-APC (BD Pharmingen, cat no. 560936), anti-CD95-APC (BD Pharmingen, cat no. 558814), anti-ki-67-PE (Ebioscience Cat no. 12-5699-42), and anti- IgD-FITC (BD Pharmingen, cat no. 555778). After staining, the cells were analyzed by 10-color flow cytometry (Navios, Beckman Coulter), with at least 20,000 CD19+ events collected for each analysis.