Detection of nonsecreted IL-4 in Th1 and Th2 clones using 8D4-8 mAb.

T-cell clones were stimulated for 5 hours by 50 ng/mL PMA and 300 ng/mL ionomycin. (A) Dual intracellular stainings of a Th1 clone using anti-IFNγ mAb and MP4 or 8D4 anti–IL-4 mAb. The right panel shows specific inhibition of 8D4 IL-4 staining by preincubation of 8D4-8 anti–IL-4 mAb with 0.1 μg/mL recombinant IL-4. (B) In the top panels, the same experiments were performed on the Th2 clone. Bottom panels, left: T-cell clones were stimulated in the presence or absence of BFA and further stained with MP4 and 8D4 anti–IL-4 mAb. Bottom panel, right: confocal analysis of MP4 or 8D4 anti–IL-4 mAb staining on the Th2 clone, after stimulation in the presence or absence of BFA. The arrow indicates a cytoplasmic cone of secretion. (C) Determination on the Th2 clone of the susceptibility to apoptosis of MP4⁺ and 8D4⁺ cells using the nuclear dye 7-AAD.