Cd39 apc

Whole blood was drawn in EDTA tubes and the cells were incubated with fluorochrome-conjugated antibodies against human CD3-FITC, HLA-DR-FITC, CD45RA-FITC, CD8-PE, CD16+56-PE, CD69-PE, CD28-PE, CD3-PerCP, CD45-PerCP, CD45RO-PerCP-Cy5.5, CD4-PE-Cy7, CD25-PE-Cy7, CD25-APC, CD39-APC, CD62L-APC, CD8-APC-H7, CD4-APC-H7, CD3-Horizon V450, CD56-Horizon V450, all from BD Biosciences (San Jose, USA). Anti-Foxp3-PE was purchased from eBioscience (San Diego, USA) and Helios-Pacific Blue from Biolegend (San Diego, USA). Lymphocyte subpopulations from peripheral blood were measured by 4- or 7-color combinations. TruCount^™ tubes (BD Biosciences) were used for assessment of absolute numbers (cells/μl) of leukocytes (CD45), and major lymphocyte populations; T lymphocyte (CD3), T helper (CD4), T cytotoxic (CD8), B (CD19) and NK (CD16+CD56) cells, as described by the manufacturer. Absolute numbers of subpopulations were derived from the major populations. Proportions of major populations were given as % of lymphocytes and proportions of subpopulations were given as % of their parent population.

To determine the percentage and the absolute count of CD3 and CD4 T cell subsets, 50 μl of whole marrow blood was stained with CD45 PerCP-Cy™5.5, CD3 FITC, CD4 PE-Cy7™, CD8 APC-Cy7, CD16 and CD56 PE, and CD19 APC monoclonal antibodies (MoAbs) (BD Multitest 6-color TBNK) in a calibrated number of fluorescent beads (Truecount, BD Parmingen). For Treg identification, 100 μl of marrow blood was incubated with a lyophilised pellet of CD45RA FITC, CD25 PE, CD127 PerCP-Cy™ 5.5, HLA-DR PE-CY™7, CD39 APC, and CD4 APC-H7 MoAbs (BD Pharmingen). Samples were processed according to the manufacturer's guidelines and acquired on a DB FACS Canto II Flow Cytometer. The absolute number (cells/μL) of positive cells was calculated by comparing cellular events to bead events using BD FACSCanto clinical software (version 3).

To determine the percentage and the absolute count of CD3 and CD4 T cell subsets, 50 μL of whole marrow blood was stained with CD45 PerCP-Cy™5.5, CD3 FITC, CD4 PE-Cy7™, CD8 APC-Cy7, CD16 and CD56 PE, and CD19 APC monoclonal antibodies (MoAbs) (BD Multitest 6-color TBNK) in a calibrated number of fluorescent beads (Trucount, BD Pharmingen). For Treg identification, 100 μL of marrow blood was incubated with a lyophilised pellet of CD45RA FITC, CD25 PE, CD127 PerCP-Cy 5.5, HLA-DR PE-CY™7, CD39 APC, and CD4 APC-H7 MoAbs (BD Pharmingen). Samples were processed according to the manufacturer's guidelines and acquired on a DB FACSCanto II Flow Cytometer. The absolute number (cells/μL) of positive cells was calculated by comparing cellular events to bead events using BD FACSCanto clinical software (version 3).