P-FPMI-139 - P-FPMI-139
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 the patient (IRAK4 Q293X mutation). 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.