Mtp 900

HEK293FT cells were seeded at a density of 8 × 10⁴ cells/well in a poly-d-lysine coated 48-well plate with DMEM containing 2% FBS. After overnight culture, cells were transfected with mock or pcDNA3.2-GPR55-V5 using PEI MAX and Opti-MEM for 24 h. GLUTag cells were seeded at a density of 8 × 10⁴ cells/well in a poly-d-lysine coated 48-well plates with DMEM containing 10% FBS and incubated for 48 h. Cells were washed with standard extracellular buffer (Hepes-NaOH, pH7.5, 143.4 mM NaCl, 4.5 mM KCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 5.5 mM glucose) containing 5 μM Fura-8AM (AAT Bioquest, Sunnyvale, CA, USA) for 15 min at 37 °C. After adaptation to room temperature (i.e., ~25 °C) for 15 min, the cells were washed three times and medium was replaced to fresh standard extracellular buffer containing 10 µM CID16020046 and incubated for 15 min, followed by stimulation of 10 μM (for HEK293FT cells) or 20 μM (for GLUTag cells) curcumin. Fluorescence intensity (Ex: 365 nm and Em: 530 nm) was determined with microplate reader (MTP-900, Corona Electric, Ibaraki, Japan) at 1-s intervals before and after stimulation with curcumin for 4 min. The integrated value of the intensity was calculated.

rGSTs of PRMs catalyze the conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB) substrate [44 (link)]. In this study, we measured enzyme activity at different concentrations of each rGST in the presence of a constant concentration of GSH and the CDNB substrate. Briefly, 20 µL of each rGST (1, 2, and 4 µg) in Tris/NaCl buffer (10 mM Tris, 0.5 M NaCl, pH 7.4) were placed in the 96-well microtiter plate in triplicate and the same amount of Tris/NaCl buffer was placed in the control wells. We dissolved 100 µL of 1 mM CDNB in substrate buffer (100 mM potassium dihydrogen phosphate, 1 mM EDTA, pH 6.5, and 2 mM GSH) and placed it in all the wells. Immediately, the absorbance at 340 nm was measured every minute using a microplate reader MTP 900 (Corona Electric Co., Ltd, Ibataki, Japan) for 20 min. To adjust the spontaneous hydrolysis of CDNB, the mean absorbance of the control wells was subtracted from that of the test wells. The specific enzyme activities (μmol/min/mg protein) at different concentrations of each rGST were calculated using the following formula [54 (link)].
$$\frac{\left(At2-At1\right)\times 1000}{A\epsilon \times \left(t2-t1\right)\times b\times m}\times Vtot$$
At1—initial time point
At2—final time point
t1—starting time (min)
t2—end time (min)
Aε—the molar extinction coefficient of CDNB (Aε = 9.6)
b—path length of the spectrophotometer (0.286 cm)
m—the quantity of rGST per well (mg)
Vtot—total volume per well (L).