Ten animals from each group were sacrificed immediately after exhaustive exercise, and 10 at 24 h after exhaustive exercise. Anesthesia was induced with 2% isoflurane inhalation at 0.8 L·min−1, maintained at 1% at 0.8 L·min−1. Blood samples were collected from the abdominal aorta using a 1-mL syringe mounted with a 20-gauge needle and coated with heparin (5000 UI·mL−1; Nipro, Osaka, Japan). Blood samples were transferred to a tube coated with heparin and centrifuged at 2600g for 10 min, then plasma was stored at −80°C until analysis.
The liver tissues were snap-frozen by immersing the samples in liquid nitrogen and stored at −80°C until analysis.
We isolated leukocytes from the bone marrow using previously described methods with some modifications (20 (link)). Femurs and tibiae were removed, and bone marrow was harvested by flushing with Hanks’ balanced salt solution without Ca2+/Mg2+, 30 mM HEPES, and 15 mM EDTA. A single-cell suspension was created by passing the suspension through a 21-gauge needle. The cells were centrifuged at 1200 rpm for 5 min at room temperature. The resultant pellet containing leukocytes was suspended in 5 mL of red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO) and centrifuged at 1200 rpm for 5 min at room temperature. Cells were resuspended in Versa Lyse (Beckman Coulter, Miami, FL).
The liver tissues were snap-frozen by immersing the samples in liquid nitrogen and stored at −80°C until analysis.
We isolated leukocytes from the bone marrow using previously described methods with some modifications (20 (link)). Femurs and tibiae were removed, and bone marrow was harvested by flushing with Hanks’ balanced salt solution without Ca2+/Mg2+, 30 mM HEPES, and 15 mM EDTA. A single-cell suspension was created by passing the suspension through a 21-gauge needle. The cells were centrifuged at 1200 rpm for 5 min at room temperature. The resultant pellet containing leukocytes was suspended in 5 mL of red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO) and centrifuged at 1200 rpm for 5 min at room temperature. Cells were resuspended in Versa Lyse (Beckman Coulter, Miami, FL).
