Adenosine

Adenosine, dopamine, histamine, and Adenosine triphosphate (ATP) were purchased from Acros Organics (Morris Plains, NJ), and H₂O₂ was purchased from Macron (Center Valley, PA). Stock solutions were prepared in 0.1 M HClO₄ to 10 mM concentration. The final working solutions were prepared by diluting the stock solution in the phosphate-buffered saline (PBS) containing 131.25 mM NaCl, 3.00 mM KCl, 10.0 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.0 mM Na₂SO₄, and 1.2 mM CaCl₂ with pH adjusted to 7.4. The buffer was also adjusted to pH 7.3 by HCl or pH 7.5 by NaOH to test the effect of pH change. In vitro experiments and electrode calibration were conducted with a flow cell connected to a syringe pump (Harvard Apparatus, Holliston, MA) and a six-port loop injector with an air actuator (VIVI Valco Instruments, Houston, TX).

Platinum wire (50 μm diameter) and glass capillary tubes were
obtained from A-M Systems (Sequim, WA, USA). Polyimide capillaries with inner
diameter of 100 μm were obtained from Polymicro Technologies (Lisle, IL,
USA). Silver conducting epoxy was obtained from MG Chemicals (Ontario, Canada)
and 5-minute epoxy was obtained from Devcon (OH, USA). Glutamate oxidase
(GlutOx) (EC 1.4.3.11, 25 U/vial; from E. coli) was obtained
from Cosmo Bio USA, Inc. (Carlsbad, California, USA). Ascorbate oxidase (AsOx)
was obtained from Alfa Aesar (Haverhill, MA USA). Silver wire, acetic acid,
L-ascorbic acid, uric acid, adenosine, dopamine, and
serotonin hydrochloride were obtained from Acros Organics (NJ, USA). Chitosan
from shrimp shells, o-phenylenediamine, albumin from bovine serum,
L-glutamic acid, and D-glucose anhydrous
were obtained from Sigma-Aldrich (St. Louis, MO, USA). Isothesia was obtained
from Henry Schein. Sulfuric acid was obtained from Fisher Scientific (Fair Lawn,
NJ, USA).

Adenosine (purity > 99%) was purchased from Acros Organics (Waltham, MA). Bovine serum albumin, fraction V, heat shock isolated was purchased from Spectrum Chemical Mfg. Corp (New Brunswick, NJ). Lipopolysaccharide (LPS) from Escherichia coli was purchased from Novus Biologicals (Centennial, CO). Fetal bovine serum and rat alveolar macrophages, NR8383 [AgC11x3A, NR8383.1] (ATCC CRL-2192), were purchased from ATCC (Manassas, VA). Penicillin–streptomycin, l-glutamine, Dulbecco’s phosphate-buffered saline (DPBS), ACK lysis buffer, Nigericin sodium salt, and endotoxin-free sterile water and HPLC grade methanol were purchased from Thermo Fisher Scientific (Waltham, MA). Antimouse IL-6 (MP5-20F3) APC antibody was purchased from BioLegend (San Diego, CA). RPMI medium was purchased from Life Technologies (Gaithersburg, MD). Rat IL-1β uncoated enzyme-linked immune assay (ELISA) was purchased from R&D Systems (Minneapolis, MN). Mouse TNFα uncoated ELISA kit, antimouse CD16/CD32 and antimouse CD86 (B7-1) PE antibodies, and buffers used in flow cytometric analyses were purchased from Invitrogen (Waltham, MA).

Adenosine was purchased from Acros Organics (New Jersey, US). A₁ (DPCPX), A_2A (ZM241385, A_2B (MRS1754), and A₃ (MRS1220) receptor antagonists were purchased from Tocris Bioscience (Minneapolis, MN). The final concentration of Adenosine used in the PEK assay was 300nM. The Adenosine receptor antagonists were diluted to concentrations that would allow for specific receptor antagonism with minimal crossover to other receptors based on previous publication (14 (link)). The following concentrations were used: A₁ (10nM), A_2A (30nM), A_2B (10nM), A₃ (10nM). These agents were used in a slightly modified PEK assay. During the ice incubation of the opsonized bacteria the thioglycollate elicited macrophages or neutrophils were incubated with Adenosine for 15 minutes in a 37°C shaker and then the PEK assay continued as described above. For the experiments utilizing Adenosine and Adenosine receptor antagonists the cells were first exposed to the antagonists for 15 minutes in the 37°C shaker and then the Adenosine was added and the cells were kept in the shaker for an additional 15 minutes.