Cd4apc clone rpa t4

The thawed PBL, allowed to rest overnight as described above, were used for an in vitro proliferation assay using the fluorescent cell proliferation indicator CytoTell green (AAT Bioquest; LuBioScience), according to the manufactures protocol (20 min incubation at room temperature in 1:600 diluted dye). Cells (150′000 in 270 μl per well) were plated in duplicates in a 96 U-bottom well plate (TTP, Switzerland) either in control medium or in stimulation medium with 2.5 μg/ml pokeweed mitogen or in stimulation medium with 0.1% phytohaemagglutinin (PHA-M, Sigma-Aldrich, Switzerland). The plates were incubated at 37 °C and 5% CO₂ in humid atmosphere. Proliferative responses of the stimulated PBL and the controls were measured after 7 days of incubation by flow cytometry using the intracellular dye dilution method and following surface marker antibodies: CD3-PerCP (clone SK7, BD biosciences), CD4-APC (clone RPA-T4, BD bioscience), CD8-PE (clone RPA-T8, BD biosciences). Cells were incubated with 5 μl of antibodies for 15 min at room temperature, washed and resuspended in 200 μl PBS. Cell fluorescence was assessed with FACScalibur flow cytometer and data were analyzed using the proliferation tool of the FlowJo, 9.0 software.

Peripheral blood samples (10–20 ml) from 20 healthy young adults (13 F/7 M, age range 22–35 years) were collected in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of whole blood over a Ficoll-Paque gradient (Biochrom) and washed 4× with ice-cold RPMI1640 culture medium (Gibco). CD19+ B cells, CD14+ monocytes, CD3+ T cells, CD4+ CD25− Th cells, CD4+ CD25+ Th cells, CD8+ T cytotoxic cells, CD4+ CD45RA+ CD25− naive Th cells, and CD4CD45RO+ CD25− memory Th cells were isolated by cell sorting using a BD FACS Aria II flow cytometer (BD Biosciences). The sorting strategy is shown in Figure S1 Supplementary Material. The isolated cell populations were phenotyped and used when their purity reached >95%. The antibodies used for cell sorting and phenotyping were the mouse antihuman monoclonal antibodies (mAbs) CD3-APC-H7 (clone SK7), CD19-APC (clone HIB19), CD14-FITC (clone M5E2), CD4-APC (clone RPA-T4), CD8-FITC (clone HIT8a), CD25-PE (clone M-A251), CD45RA-APC-H7 (clone 5H9), and CD45RO-PE-Cy7 (clone UCHL1) (BD Biosciences), CD25-PC5 (clone B1.49.9) (Beckman Coulter). Fluorescence minus one controls were used to identify any background spread of fluorochromes and establish gating boundaries. The data were analyzed using the BD FACS DIVA software v.8.