Leukocyte phenotyping has been described in a recent publication by our group.20 (link) Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral whole blood by Ficoll-Paque density gradient centrifugation, and the pellet was re-suspended with a dilution of 1×106 cells/mL in Fluorescence-Activated Cell Sorting (FACS) buffer. 100μL suspension was then distributed in 4 tubes:

Tube 1: not stained, containing 100μL of the PBMC suspension.

Tube 2: for CD3 assessment: 100μL of the PBMC suspension, Monoclonal antibodies (3μL CD3 FITC - BD Bioscience 561,806 + 40μL FACS Buffer);

Tube 3: for T and B cell phenotyping; 100μL of the PBMC suspension, Monoclonal antibodies (3μL CD3 FITC + 3μL CD4 PE - BD Bioscience 555,347 + 2μL CD8 PE-Cy7 - BD Bioscience 557,746 + 3μL CD19 PE-Cy5 - BD Bioscience 555,414);

Tube 4: for monocytes (M1, M2) and natural killers (NK) phenotyping: (3μL CD3 FITC + 2μL CD56 PE-Cy7 - BD Bioscience 557,747 + 3μL CD16APC - BD Bioscience 561,248 + 2μL CD14APC-Cy7 - BD Bioscience 561,384 + 3μL HLA-Dr PerCP - BD Bioscience 347,402).

Incubation for staining was carried out at 4°C for 30 minutes and the tubes were subsequently washed with 2mL FACS buffer, spinned at 1500rpm × 5min, dechanted, and re-suspended in 500μL of FACS buffer.
The tubes were analyzed with FACScanto 2 BD USA and with BD FACSdiva v8.1.