Heparinized hematocrit tube

For the toxicity assessment of GTN057 in vivo, we intraperitoneally (i.p.)‐injected male 5‐week‐old ICR mice (CLEA, Tokyo) with GTN057 (0, 50, or 100 mg/kg) dissolved in 1% Tween® 80 + 10% DMSO in saline on 2 consecutive days of every 3‐day period for 2 weeks. The mice were weighed every week. Peripheral blood was collected with a heparinized hematocrit tube (Terumo, Tokyo) from the tail vein of each mouse every week and was stained with Türk's solution (Merck). The numbers of leukocytes and neutrophils were counted using light microscopy.

The SW-experienced medaka and control FW medaka were transferred directly to SW, and the surviving fish were sampled at 0 h (FW), 3 and 6 h after SW transfer. After anesthesia in 0.05 % (v/v) 2-phenoxyethanol (Wako, Osaka, Japan), the blood (less than 6 μl) was collected from the heart with a glass capillary. The collected blood was immediately transferred into a heparinized hematocrit tube (Terumo, Tokyo, Japan), which was sealed to prevent evaporation. The blood osmolality was measured as previously described [13 (link), 14 (link)]. In brief, approximately 4 μl of the blood was applied onto a sample disc (6 mm in diameter) made of #1 filter paper (Whatman, Kent, UK), and the osmolality was measured with a vapor pressure osmometer (model 5520, Wescor, Logan, UT) on the same day of blood sampling. For the measurement of plasma Na⁺ and Cl⁻ concentrations, the collected blood in a heparinized hematocrit tube was centrifuged at 10,000 × g for 10 min at 4 °C. One microlitre plasma was collected from the centrifuged blood sample with 1-μl Microcaps (Drummond scientific, Broomall, PA), and diluted with 1 ml of Milli-Q water. Plasma Na⁺ and Cl⁻ concentrations were determined with an ion analyzer (IA-200, TOA-DKK, Tokyo, Japan).