Fl600 dual band plate reader

For the UGT method, primary hepatocyte cells were incubated with 150 µM estrone (for UGT1A3) and 600 µM 1-naphthol (for UGT1A6) in presence of UPF 14 µg or 285 µg/mL and FVF 285 µg or 1300 µg/mL. Fucoidan was compared to control β-naptholflavone 80 µM. The cultures were maintained in duplicate for each experimental time point, including 2 hours, 6 hours, and 24 hours. To determine UGT induction, remaining substrate concentrations in samples were measured with FL600 Dual-Band plate reader from BioTek Instruments, Inc using UV detection at 230 nm for estrone (UGT1A3) and at 280 nm for 1-naphthol (UGT1A6).

UGT activity was evaluated following a method described by Liu and Franklin.^{15 (link)} Inhibition assays were carried out in 200 µL total volume, 96-well UV-Vis plates. The 3 primary isoforms of UGT associated with drug metabolism were evaluated: UGT1A3, UGT1A6, and UGT2B17. Substrates for each isoform included estrone (UGT1A3), 1-napthol (UGT1A6), and testosterone (UGT2B17). In short, the reaction of 2 mM uridine 5′-diphosphoglucuronic acid (UDPGA) with each respective substrate in the presence of 2 µL human liver microsomes (20 mg/mL), diluted in 50 mM potassium phosphate buffer (pH 8.0), was measured by UV absorbance on a FL600 Dual-Band plate reader (BioTek Instruments, Inc) at either 220 nm (UGT1A6) or 230 nm (UGT1A3 and UGT2B17). UPF or FVF was added as the test/experimental agents using either UPF 14 µg or 285 µg/mL or FVF 285 µg or 1300 µg/mL and serially diluted 1:3 for 8 wells. Removal of UDPGA was used as a negative control.

QOR activity was evaluated using the method described by Benson and colleagues.^{16 (link)} Assays were carried out in 96-well UV-Vis plates with total volume of 200 µL per well. The reaction mixtures contained 0.7 mg of bovine serum albumin, 25 mM Tris-HCl (pH 7.4), 0.2 mM NADPH, 40 µM 2,6-dichlorophenolindophenol (DCPIP), and control inhibitor, dicoumarol 10 mM. As with the UGT assay, UPF or FVF was added as the test/experimental agents using either UPF 14 µg or 285 µg/mL or FVF 285 µg or 1300 µg/mL, then serially diluted 1:3 for 8 wells. NADPH was measured by UV absorbance at 200 nm or DCPIP at 600 nm on a FL600 Dual-Band plate reader from BioTek Instruments, Inc.