Stimulation and Incubation of Whole Blood Samples for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

To begin, prepare 2.7 milliliter blood collection tubes without anticoagulant, with antigens and co-stimulatory antibodies under sterile conditions. Add anti-CD28 and anti-CD49d into each tube, including the negative control. If frozen, thaw the ready-to-use stimulation tubes at room temperature about 30 to 60 minutes before whole blood stimulation.

Collect venous blood from the donor or patient using blood collection tubes with lithium heparin anticoagulant. Pipette the required amount of heparinized whole blood into the stimulation tubes under a sterile workbench. Carefully invert the tubes about 5 to 10 times.

Then, place the tubes in an incubator at 37 degrees Celsius. After four hours of incubation, prepare one milligram per milliliter brefeldin A solution in RPMI medium. In a sterile workbench, uncap the tubes used for flow cytometric analysis.

Then transfer 1%of the sample volume of the brefeldin A solution into the stimulation tube. Incubate the capped tubes at 37 degrees Celsius for another 16 to 18 hours.