Himac cs150gxii

Exosomes were prepared using sequential centrifugation according to methods described previously. The exosomes were isolated from the supernatants of CD133 + /Lin-/CD45- cells using ExoQuick-TC Kit (System Biosciences, USA) in accordance with manufacturer’s instructions. The CD133 + /Lin-/CD45- cells were cultured in a conditioned medium containing 10% exosome-free fetal bovine serum (FBS) for 48 h for the preparation of exosomes isolation, and then the supernatants of CD133 + /Lin-/CD45- cells were collected by initial centrifugation at 3,000 g for 15 min to pellet and remove the cell debris, then followed by sequential centrifugations at 13,000 g for 30 min, followed by centrifugation at 100,000 g for 60 min (Himac CS150GXII, HITACHI, Japan). Then the exosome-enriched fraction was resuspended in phosphate-buffered saline (PBS). To identify the Exos, exosomes resuspended in 0.1 mL of PBS was used for a transmission electron microscope and particle size analysis, and exosomes resuspended in 0.1 mL RIPA buffer for protein quantification and Western blotting. 0.5 × 10⁹ exosomes were administered to cultured cells in vitro and 5 × 10⁹/40μL to experiment rats in vivo to evaluate the function of Exos. Besides, the morphologies of exosomes were observed with a transmission electron microscope (Hitachi H-7100 microscope; HITACHI, Japan).

Midguts (n = 5) of shrimp intestine from each group were homogenized (IKAT 25, Staufen, Germany) for 1 min (5000 × g) in cold distilled water and centrifuged (Himac CS150GXII, Hitachi, Tokyo) for 20 min (8,000 × g) at 4 °C. The supernatant was used to measure the digestive enzyme activities. Protease activity was determined by the casein-hydrolysis method of Furne et al.^{53 (link)}. Amylase activity was determined by the starch-hydrolysis method of Zokaeifar et al.^{54 (link)}. Lipase activity was determined according to the method of Muralisankar et al.^{19 (link)} by degrading triacylglycerol to free fatty acids. Digestive enzyme activities are expressed as U/mg of protein.
The concentration-response curves between T-2 and digestive enzyme activities in shrimp intestine were constructed using Origin 8.5. The curves were drawn with T-2 concentration (mg/kg) as the x-axis, and the ratio between the value of the experimental group and the control group (relative coefficient) as the y-axis. The NOAEL (no observable adverse effect level, concentration of T-2 when the ratio of enzyme activity was 1), MEC (maximal effect concentration of T-2, concentration of T-2 when the ratio of enzyme activity was maximum) and EC₅₀ (concentration for 50% of maximal effect, concentration of T-2 when the ratio of enzyme activity was 0.5) were calculated by GraphPad Prism 7 (GraphPad Software, La Jolla, CA).