mRNA expression analysis

Strain specific A2AR expression analysis in homozygous B-hA2AR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry with species-specific anti-A2AR antibody. Mouse A2AR was detectable in WT mice, human A2AR was detectable in homozygous B-hA2AR due to the anti-mouse A2AR antibody also cross-reacts with human A2AR.

Strain specific A2AR expression analysis in homozygous B-hA2AR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry with species-specific anti-A2AR antibody. Mouse A2AR was detectable in WT mice, human A2AR was detectable in homozygous B-hA2AR due to the anti-mouse A2AR antibody also cross-reacts with human A2AR.

cAMP measurements in spleen and bone of wild-type mice and B-hA2AR mice incubated with different concentration of CGS21680 (an A2AR agonist), and analyzed by LANCE® Ultra cAMP Kit (PerkinElmer). The cAMP levels of spleen cells and bone marrow cells in B-hA2AR mice were higher than those in C57BL/6 mice at 500μM CGS21680. It was speculated that B-hA2AR mice were more sensitive to the stimulation of CGS201680 than wild mice. At the background level, there was no significant difference in A2AR mediated cAMP pathway between B-hA2AR and C57BL/6 mice. Compared with C57BL/6, B-hA2AR more effectively mediated the A2AR-dependent cAMP pathway under strong stimulation.

Analysis of blood leukocyte subpopulations in B-hA2AR mice

In vivo efficacy of A2AR small molecule drugs