P-FPMI-135 - P-FPMI-135
PBMC were incubated for 4 hrs at 37 degree Celsius in 5% CO2 in 100 ng/ml recombinant human TNF-alpha. The reagent was tested for endotoxin and reconstituted in endotoxin-free water. Stimulation was performed using PBMC from 5 healthy adults. PBMC were transferred to 15 ml tubes (Falcon; Becton Dickinson), using 1ml of Versene to detach adherent cells. PBMC were washed twice into PBS containing 5 mM EDTA and 2% FCS to a final concentration of ~1 x 10^7 cells/ml. Cells were rotated end over end at 4 degree Celsius for 1 hr with 25 ul of anti-CD14 conjugated magnetic beads (M450; Dynal; Invitrogen) per ml of cells. Beads were immobilized with a magnet (Dynal; Invitrogen) and washed 3 x 1 ml with PBS. RNA was isolated from CD14+ PBMC with RNeasy Mini kit, treated with RNase-Free DNase (Qiagen Inc., Canada) and eluted in RNase-free water (Ambion Inc., Austin, TX, USA) as per manufacturer's instructions. An equal quantity of RNA from each of the healthy individuals was pooled into one control sample for the microarray.