Edta microtainer tube

Rats were anesthetized and bled into pre-chilled EDTA-microtainer tubes (Becton­Dickinson). The blood samples were processed to plasma which was stored in labeled 1.5-ml Eppendorf tubes at -80°C. Plasma samples were thawed to room temperature and assayed by ELISA for IL-6, IL-β and TNF-α (R&D Systems).

350–400 μL of capillary blood was collected into EDTA microtainer tubes (Becton Dickinson, New Jersey, United States) by finger prick. For RNA preservation, 100 μL of whole blood was transferred to a tube containing 500 μL of RNAlater (Sigma-Aldrich, Missouri, United States) within 2 h of collection and stored at − 80 °C until RNA extraction [39 , 40 (link)]. The remaining blood was centrifuged, plasma removed and stored at − 20 °C. The red cell pellet was stored at − 20 °C until DNA extraction.

Female C57BL/6J mice (6–8 weeks old) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The murine pulmonary fibrosis model was established by delivering bleomycin solution to the lungs of mice with a MicroSprayer aerosolizer (IA-1C; Penn-Century, PA, USA) inserted into the trachea. Briefly, mice received a single dose of bleomycin (Bleocin, Nippon Kayaku, Japan) in 50 μl of sterile phosphate-buffered saline (PBS) by intratracheal spray under anesthetization. At day 7 after intratracheal spraying, the mice were randomized into three groups without blinding to receive vehicle, 60 mg/kg nintedanib, or 50 mg/kg SC-43 via oral gavage until the end of the experiment. Blood samples were obtained by cardiac puncture in EDTA microtainer tubes (BD Biosciences, NJ, USA) and sent to the National Laboratory Animal Center for complete blood count. The lungs were lavaged twice with 1 ml cold sterile saline through a plastic cannula. The bronchoalveolar lavage fluid (BALF) was centrifuged at 300 × g, 4 °C for 5 min. The cell pellets were resuspended in the cell staining buffer (2% FBS and 0.02% NaN3 in PBS) for flow cytometry analysis. All experimental procedures using these mice were performed according to protocols approved by the Institutional Laboratory Animal Care and Use Committee of Cardinal Tien Hospital (IACUC-108A-001 and IACUC-110C-005).