Figure 4.

Importance of Using the Correct Beads for Compensation Controls

Example generated from cryopreserved PBMC stimulated in vitro with anti-CD3 (clone UCHT1, Bio-Rad, Hercules, CA) and CD49d/CD28 co-stimulatory antibodies (clones L293 and L25, BD Biosciences) for 6 h at 37°C, 5% CO₂. Following 1 h of stimulation, monensin (0.7 μg/mL final concentration; BD Biosciences) and brefeldin A (1 μg/mL final concentration; Sigma-Aldrich, St. Louis, MO) were added, and cells were incubated for 5 h more. Cells were stained to quantify cytokine production. The example shows the expression of TNF-α (TNF-α PE Cy7 clone Mab11, eBioscience) and IFN-γ (IFN-γ Alexa Fluor 700, Invitrogen) in memory CD8+ T cells.

(A) Compensation control prepared with Plus beads.

(B) Representative example of stimulated memory CD8+ T cells. In A and B, the orange selection indicates the events above median of the compensation peak, which would not be compensated correctly.

(C) Compensation control prepared with normal beads.

(D) Representative example of stimulated memory CD8+ T cells. In C and D, the purple selection indicates the events above the compensation peak, which would not be compensated correctly.