Ultima gold scintillation cocktail

Barley BG (Megazyme) was added to water and brought to 90°C whilst stirring continuously on a magnetic stirrer until the mixture was clear. The mixture was cooled to room temperature and diluted to 2, 4, and 6 mg / mL with standard MBS buffer. Two types of barley BG were used namely a) BG with an average molecular weight of 650,000 (BG-HMW) and b) BG with an average molecular weight of 229,000 (BG-LMW). Glucose uptake studies were conducted in MBS transport buffer containing 125 pmol radiolabeled glucose and BG (0, 1, 2, and 3 mg / ml). Oocytes (15 (link)) were transferred to 200 μl transport buffer and incubated for 30 min at room temperature. Transport was terminated by adding ice cold MBS in excess. The oocytes were washed four times with ice cold MBS and lysed individually in vials containing 200 μl sodium dodecyl sulfate (10%). Ultima Gold scintillation cocktail (5 ml) (PerkinElmer) was added to each vial and internal radioactivity was quantified by liquid scintillation spectrometry as counts per minute (CPM). [³H] 2-deoxyglucose and [³H] 3-O-methyl-D-glucose were used as substrate in glucose transport experiments involving oocytes expressing GLUT2 and SGLT1, respectively.

Dulbecco’s modified Eagle’s medium, Flp-In CHO cells, and hygromycin B were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from ThermoTrace (Melbourne, VIC, Australia). [³H]spiperone and Ultima Gold scintillation cocktail were from PerkinElmer (Boston, MA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). All compounds were synthesized as described previously^{2 (link),3 (link)} with the exception of MIPS1868 and MIPS1726, which were synthesised as described in supplementary information.

Transport activity for [³H]riboflavin across the placenta of pregnant Slc52a3+/− mice, which were mated with Slc52a3+/− male mice, at gestation date 16.5 was determined as previously reported36 (link). Pregnant dams were anesthetized with an intraperitoneal administration of sodium pentobarbital. Surgery was performed on each animal lying on a heating pad to maintain constant body temperature. Then, radioisotope-labeled riboflavin (500 nM of [³H]riboflavin, 0.903 TBq/mmol, Moravek Biochemicals) or glucose (40 μM of D-[U-¹⁴C]glucose, 11.5 GBq/mmol, GE Healthcare) was administrated as a bolus via the femoral vein (10 mL/kg). Five minutes after administration, fetuses and placentas were removed and weighted. Fetal tails were collected, and used for genotyping as described above. Fetuses were homogenized in saline, and the samples were placed into Ultima Gold scintillation cocktail (PerkinElmer) followed by solubilization in SOLVABLE (Packard Instrument Co.). The radioactivity was measured by liquid scintillation counting, and the levels of [³H]riboflavin and D-[U-¹⁴C]glucose in each fetus were determined and their transporter activities were calculated.