Deoxycoformycin dcf

Adenosine levels in bronchoalveolar lavage fluid (BALf) were measured as previously described (Wakamiya et al., 1995 (link)). Briefly, to measure the nucleoside levels in BALF, mice were anesthetized with 2.5% avertin and the lungs were lavaged 4 times with 0.3 ml PBS containing 2 μM dipyridamole (Sigma-Aldrich, St. Louis, MO, USA) and 5 μM ADA-inhibitor deoxycoformycin (dCF, R&D Systems Inc, Minneapolis, MN, USA), which pooled 1.0 ml BALF. The BALF was then centrifuged to remove cells and debris. To measure nucleoside levels, 200 μl BALF supernatant were loaded to the HPLC meter per reading and the flow rate was set at 1 ml/min. The representative peaks were identified and quantitated by running known external standard curves.

Bronchoalveolar lavage fluid (BALF) was obtained as described previously (Wakamiya et al. 1995 (link)). Briefly, at time of sample collection, mice were anesthetized with avertin and blood was collected and centrifuged to isolate plasma. Lungs were lavaged 4 times with 0.3 mL PBS containing 10 μmol/L dipyridamole (Sigma–Aldrich, St. Louis, MO), 10 μmol/L ADA‐inhibitor deoxycoformycin (dCF; R&D Systems Inc, Minneapolis, MN), and 10 μmol/L αβ‐methylene ADP, which pooled approximately 1.0 mL fluid. A sample was separated for cell count determination using a hemocytometer. The remaining BALF was centrifuged and supernatant and cell pellet were stored for further analyses. Protein concentration in BALF was determined using Bio‐Rad protein assay (Bio‐Rad, Hercules, CA) with BSA standards. After lavage, the pulmonary vasculature was perfused with PBS, lungs were inflated with constant pressure of 20 cm H₂O and fixed in formalin for 24 h. Lungs were processed and paraffin embedded. Five micrometer sections were cut and used for histology and immunohistochemistry.