Celltac

The obtained BALF was centrifuged at 400× g at 4 °C for 15 min, and the supernatant was transferred to a new tube and frozen for measuring the cytokines. The pellets were washed by suspension with polymorphonuclear leukocyte (PMN) buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 5.6 mM C₆H₁₂O₆) and centrifuged at 400× g at 4 °C for 15 min. After the supernatant was removed, the pellets were re-suspended with 1 mL of PMN buffer. The number of cells in the BALF was counted by Celltac (Nihon Kohden Corp., Tokyo, Japan), and the cells were splashed on a slide glass using cytospin. After the cells were fixed and stained with Diff-Quik (Sysmex Corp., Hyogo, Japan), the number of neutrophils and alveolar macrophages was counted by microscopic observation. These data have been published in our previous reports [22 (link),23 (link),25 (link),26 (link),27 (link),28 (link),29 (link)].

All animal experiments were performed in accordance with protocols approved by the Animal Experiment Review Board of the University of Miyazaki. Ramp1-deficient mice were described elsewhere^{31 (link)}. C57BL/6 CD45.1 congenic mice (B6-CD45.1) were purchased from Sankyo-Lab Service (Tokyo, Japan). For analysis of blood counts, PB from the tail vein was collected in EDTA-coated microtubes and analyzed with a hematology analyzer (CellTac, NIHON KOHDEN, Tokyo, Japan).

The obtained BALF was centrifuged at 400 g at 4 °C for 15 min, and the supernatant was transferred to a new tube and frozen for measuring the cytokines. The pellets were washed by suspension with polymorphonuclear leukocyte (PMN) Buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na2HPO4, 1.5 mM KH2PO4, 5.6 mM C6H12O6) and centrifuged at 400 g at 4 °C for 15 min. After the supernatant was removed, the pellets were re-suspended with 1 mL of PMN Buffer. The number of cells in the BALF was counted by Celltac (Nihon Kohden Corp., Tokyo, Japan), and the cells were splashed on a slide glass using cytospin. After the cells were fixed and stained with Diff-Quik (Sysmex Corp., Hyogo, Japan), the number of neutrophils and alveolar macrophages were counted by microscopic observation.

Animals were decapitated under general anesthesia to evaluate hematology, clinical chemistry, and histopathology parameters. Blood samples were taken for clinical chemistry tests. Total leukocyte count (WBC), erythrocyte count (RBC), platelets (Plt), hemoglobin (Hgb), hematocrit (Hct), mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), neutrophils, lymphocytes, eosinophils, and monocytes were measured using an animal blood counter (Celltac; Nihon Kohden, Tokyo, Japan). Plasma urea nitrogen (BUN), creatinine (Cr), sodium (Na), potassium (K), chloride (Cl), bicarbonate (HCO³⁻), calcium (Ca), magnesium (Mg), lactate (Lac), osmolarity (Osm), and glucose (Glu) were determined using CCX System (CCX WB; Nova Biomedical, USA). Plasma alkaline phosphatase (ALP), albumin (ALB), total bilirubin (T.Bil), direct bilirubin (D.Bil), gamma-glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) were also measured (Autoanalyser Model Biotecnica, BT 3500, Rome, Italy). In addition, liver, kidney, brain, lung, spleen, and heart tissue samples were fixed and preserved in 4% buffered formaldehyde for at least 24 h. Tissue blocks were prepared and evaluated for histopathological changes.

Blood samples were taken at 0, 3, 7, 14, 21, and 30 days after radiation under general anesthesia. The hematological and biochemical parameters were measured by using an animal blood counter (Celltac; Nihon Kohden, Tokyo, Japan), a CCX System (CCX WB; Nova Biomedical, USA), and an Autoanalyser System (Autoanalyser Model Biotecnica, BT 3500, Rome, Italy)^{36 (link)}.