|Values are valid only on day of printing.|
Peripheral blood mononuclear cells (2x10 cells/mL) in RPMI 1640 medium supplemented with L-glutamine and 20% human AB serum, are added to wells of a sterile, flat-bottom, 48-well culture plate that contains either medium plus 20% AB serum alone (unstimulated) or varying concentrations of antigens: Candida albicans (1, 10, and 20 micrograms/mL) and tetanus toxoid (0.5, 1, and 2.5 micrograms/mL). Cells are analyzed by flow cytometry for day 0 viability as outlined below. After 6 days of incubation, EdU (thymidine analog) is added to all wells, where it becomes incorporated into the synthesizing DNA during a final 18-hour to 24-hour incubation period. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.
On day 7 following the second incubation, the 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 T cells. Results are reported for the percent viable cells on day 0, as well as percent proliferating cells within each group of lymphocytes and T cells.(Package insert: 4; Invitrogen Click-iT-EdU; unpublished Mayo method)
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Do not send specimen after Thursday.