Skip to main content
. Author manuscript; available in PMC: 2016 Aug 15.
Published in final edited form as: J Immunol. 2015 Jul 1;195(4):1732–1743. doi: 10.4049/jimmunol.1402288

Figure 1. Overview of the experimental protocol and tissue-specific Adora2b-deficient mice.

Figure 1

(a) Experimental protocols used. Model 1. Myocardial ischemia consisted of 60 minutes of ischemia followed by 2 h of reperfusion. Model 2. For ischemic preconditioning studies, 4 cycles of 5 min ischemia and 5 min reperfusion prior to the onset of myocardial ischemia was performed. (b) Overview of tissue-specific Adora2b deletion using a Cre-lox-system. (c) Genotyping data. Tissue was collected from the organs as labeled and genotyped using conventional PCR (GeneTyper, NY, USA). Genotyping analyzed for Adora2b floxed (lox), Cre-recombinase (Cre), Adora2b wildtype (WT) and for the Adora2b knockout (KO) signal. Left, representative PCR results from heart or aortic tissue from Adora2bf/f -Myosin-Cre+ mice (cardio-myocyte specific); note: the positive KO band in cardiac tissue, in contrast to only WT band present in the aortic tissue. Middle, representative PCR result from Adora2bf/f-VE-Cadherin-Cre+ mice (endothelial specific); note: the positive KO signal in heart and aorta. Right, representative PCR results from Adora2bf/f-Lyz2-Cre+ mice (bone-marrow derived cell specific); note: positive KO and WT band in heart and aorta. The Cre signal for Myosin-Cre+ or VE-Cadherin-Cre+ was different than the signal from Lyz2-Cre+ as a different PCR strategy was used (according to Jackson Lab protocols); Adora2b flox signal: 200 base pairs (bp); Adora2b KO signal: 366 bp, Adora2b WT signal: 1600 bp, Myosin-Cre+ and VE-Cadherin-Cre+ signal: double band at 270 and 497 bp, Lyz2-Cre+ signal: 260 bp; ladder used is depicted to the right. (d–f) RT-PCR data showing Adora2b mRNA transcript levels from the respective tissues of the tissue specific- Adora2b deleted mice. (d) Cardiomyocytes were isolated from Myosin Cre+ and Adora2bf/f -Myosin-Cre+ mice and cultured overnight, (e) endothelia and (f) myeloid cells were isolated via positive selection (CD31+ for endothelia from VE-Cadherin-Cre+/ Adora2bf/f -VE-Cadherin-Cre+ hearts and CD11b+ for myeloid cells from Lyz2-Cre+/ Adora2bf/f -Lyz2-Cre+ hearts) using magnetic beads (EasySep) and bone marrow cells were isolated from the Lyz2-Cre and Adora2bf/f -Lyz2-Cre femurs.