|Values are valid only on day of printing.|
Peripheral blood mononuclear cells (500,000 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 mitogens pokeweed (PWM) (0.5, 5, and 10 micrograms/mL) and phytohemagglutinin (PHA) (0.5, 2.5, and 5 micrograms/mL). 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, 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, CD69+ activated cells, and CD19+ B cells (PWM only). Results are reported for the percent viable, dead, and apoptotic lymphocytes, as well as percent proliferating cells within each group of lymphocytes, T cells, and B cells (PWM only).(Package insert: 4, Invitrogen Click-iT-EdU; unpublished Mayo method)
Monday through Friday
Do not send specimen after Thursday.