Infinite 2000 pro

The FSW device (PiezoWave) was purchased from Richard Wolf GmbH (Knittlingen, Germany). A 500 kHz × 64 mm focused piezoelectric transducer (H-104G) and its fundamental and third harmonic resonance impedance matching network were acquired from Sonic concepts, Inc. (Washington, United States). A RF Power Amplifier (40AD1) was obtained from Amplifier Research Inc. (Pennsylvania, United States). A RF multifunction power meter (4421) and its directional power sensor (4025) were procured from Bird Technologies Co. (Ohio, United States). A function/arbitrary waveform generator (33120A) was purchased from Agilent Technologies, Inc. (California, United States). An oscilloscope (LT354ML) was obtained from LeCroy Co. (New York, United States). An immersion planar ultrasound transducer (1 MHz, A392S-SU) and manually controlled ultrasound pulser-receivers (5072PR) were acquired from Olympus Co. (Tokyo, Japan). A slide scanner (Ventana Dp200) and its software (Ventana Image Viewer v3.2) were obtained from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). A microplate reader (Infinite 2000 Pro) and its software (i-control) were procured from Tecan Austria GmbH Co. (Grodig, Austria).

The hepatic levels of triglyceride and total-cholesterol were determined using commercial kits (Applygen, Beijing, China). All of the experiments were performed according to the manufacturer’s instructions, and the results were measured using a microplate reader (Infinite 2000 PRO, TECAN, Switzerland).

Intracellular ATP and fresh liver tissue homogenate ATP contents were detected by the ATP Assay Kit (Beyotime, Shanghai, China) according to the instructions. The optical density was detected by a multifunctional microplate reader (Infinite 2000 pro, TECAN, Switzerland). The protein content was detected to normalize the ATP level.

Cell viability was measured using the Cell Titer 96 Aqueous Solution (Promega). In brief, cells were culture in 96‐well plates and treated with RPE for 24 hr. The 100 mg/ml of RPE stock sample was treated to the cultured media as indicated concentration (50–1,000 μg/ml). Following the incubation period, 20 μl MTS solution was added, and cells were further incubated for 1 hr. Cell viability was measured with absorbance at 490 nm using a microplate reader (Infinite^®2000 PRO, Tecan, Switzerland).

The total content of chlorophyll and carotenoids was determined by the method of Wellburn and Lichtenthaler (1984) . 100 mg of fresh plant tissue was used and macerated with liquid nitrogen in 1 mL of cold 80% acetone. The mixture was centrifuged at 13,000 rpm for 10 min and the supernatant was recovered. Each sample was diluted 1:10 and absorbance was measured at 470 nm, 649 nm, and 665 nm in a microplate reader spectrophotometer (TECAN, Infinite2000pro, Austria). Chlorophyll concentration was determined using Eqs. (1) and (2), and the ratio between chlorophyll was determined using Eq. (3). Carotenoids concentration was determined using Eq. (4)

Antioxidant activity of hydroalcoholic extracts was measured by the bleaching of 1,1-Difenil-2-Picril-Hidrazil (DPPH) cation radical (Brand-Williams et al., 1995 ). A reaction mixture with 100 μL DPPH 100 mM (A517 = 0,7 − 0,8) and 10 μL protein extract was prepared. Absorbance at 515 nm was measured for 10 min at 37°C (TECAN, Infinite2000pro, Austria). The antioxidant activity were expressed as DPPH consumption percentage (%), where DPPH 100 mM was used as a control reference (Naik et al., 2005 (link)).

Utilizing a Cell Counting Kit-8 (CCK-8) we assessed cell proliferation (Bioss, Beijing, China). Transfected KIRC cells were seeded at a density of 2000 cells per well in 96-well plates and grown at 37 degrees Celsius and 5% carbon dioxide. At 0, 24, 48, and 72 hours, the CCK-8 reagent was added, and the samples were incubated for 2 hours. At 450 nm, the absorbance of each sample was measured using a microplate reader (Infinite 2000 PRO, TECAN, Switzerland).