Blocking antibody for A2tR fails to reduce A2t-dependent cytokine induction in human macrophages. (A) Seven-day-old macrophages were pretreated with either anti-A2tR–blocking antibody or rabbit polyclonal isotype control (10 μg/mL for 20 minutes at room temperature), followed by treatment with 2nM A2t for 1 hour at 37°C. RNA was isolated and reverse transcribed, and levels of the indicated species were analyzed by real-time polymerase chain reaction. (B) Monocytes were plated and aliquots were stained for A2tR expression at indicated times throughout macrophage differentiation. Histograms shown are representative of 4 donors. Black (filled) histograms = rabbit IgG isotype control, gray (open) = anti-A2tR–blocking antibody (both used at 1 μg/mL, then stained with 1:200 Alexa 488 donkey anti–rabbit). (C) Monocytes/macrophages were treated with 5nM A2t for 1 hour at 37°C at the indicated times (days after plating) during differentiation. mRNA levels were analyzed as in (B). Macrophages from the same donors were stained with goat anti–human CD64 antibody, followed by Alexa 488 donkey anti–goat secondary (values for isotype controls subtracted) at the same times to monitor macrophage differentiation. Results (A,C) are the mean ± SEM of 3 experiments with separate donors.