|Values are valid only on day of printing.|
Peripheral blood mononuclear cells (1 x10 cells/mL) in RPMI 1640 medium supplemented with L-glutamine and 5% human AB serum are added to wells of a sterile, flat-bottom, 48-well culture plate that contains either medium plus 5% AB serum alone (unstimulated) or varying concentrations of anti-CD3 antibody (aCD3) or anti-CD3 plus anti-CD28 antibodies (aCD28) in a 1:2 concentration or anti-CD3 costimulated with exogenous interleukin-2 (IL2). Cells are incubated for 72 hours, after which EdU (thymidine analog) is added to all wells where it becomes incorporated into the synthesizing DNA during a second incubation of 18 to 24 hours. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.
Following the second incubation, cells are stained for proliferation via a copper-catalyzed click chemistry reaction where the EdU, an alkyne, is covalently bonded to a fluorescent azide. Cells are also stained for the following markers: CD45+ lymphocytes, CD3+ T cells, and CD69+ activated cells. Results are reported for the percent viable lymphocytes on Day 0, as well as percent proliferating cells within each group of lymphocytes and T cells for each stimulant within the panel.(Package insert: 4, Invitrogen Click-iT-EdU; unpublished Mayo method)
Monday through Friday
Do not send specimen after Thursday.