This is the protocol we follow when testing our PBMC for reactivity to recall antigens.
- Thaw cells following the recommended procedure. Adjust cell concentration to 2.5 x 106 per mL in X-VIVO 15 medium.
- Add cells to a round bottom, 96 well plate at 100 ul of cells per well. A minimum of 15 wells should be set up to allow for all controls and antigens to be run in triplicate.
- Prepare dilutions of antigens and mitogens in X-VIVO 15 medium. Tetanus toxoid and CMV antigens should be diluted to 2 ug/ml. LPS should be diluted to 200 ng/mL. PHA should be titrated to determine optimal concentration, see note below.
- Add antigens to plate at 100 ul per well in triplicate.
- Add 100 ul of medium to each of 3 wells as a control.
- Incubate at 37°C, 6% CO2 for 4 days. The day that the culture is begun is considered day 0.
- On day 4, carefully remove 150 ul of medium from each well. Avoid removing cells.
- The cytokine in the culture medium can be assayed for interferon gamma or TNF alpha (or both) using commercially available ELISA kits. Store culture supernatants at or below minus 20°C until assay.
- We have found that 1 ug/mL of PHA is optimal, but this concentration can vary depending on the source of PHA. We recommend you titrate the concentration of PHA to determine the optimal concentration for your laboratory.
- Other media can be used instead of X-VIVO 15. RPMI 1640 supplemented with 5% fetal calf serum is one alternative. Higher background (control) values may be observed with medium supplemented with FBS.
- Dilutions of the culture medium are recommended for measurement of cytokines by ELISA. PHA stimulation will result in nanogram per mL concentrations of TNF alpha and interferon gamma. LPS stimulation will also generate nanogram per mL concentration of TNF alpha, but production of IFN gamma is donor dependent and may be negative.
- Day 4 is not the optimal time for measuring cytokines for all of the antigens and mitogens in this assay. We have chosen it for convenience rather than for peak cytokine concentration.
- Production of cytokines following stimulation with CMV antigens does not necessarily correlate with serologic testing of the donor.