The measurements of TPC and TF contents were performed in microplates (Infinite M1000 Pro Multimode Microplate Reader; Tecan Männedorf, Switzerland) according to the procedure described previously by Horszwald and Andlauer [41 (link)]. The results were calculated as milligram (mg) of gallic acid equivalent (GAE) per gram of sample for TPC, and as mg of quercetin equivalent (QE) per gram for TFC.
Infinite m1000 pro multimode microplate reader
Manufactured by Tecan
Sourced in Switzerland
The Infinite M1000 Pro Multimode Microplate Reader is a versatile laboratory instrument designed for various analytical applications. It is capable of performing absorbance, fluorescence, and luminescence measurements on microplates. The reader can detect a wide range of wavelengths and offers multiple detection modes to accommodate different experimental requirements.
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6 protocols using infinite m1000 pro multimode microplate reader
1
Quantification of Phenolics and Flavonoids
2
Alkaline Phosphatase Activity Assay of mESCs
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Alkaline phosphatase activity of mESCs was measured using the Alkaline Phosphatase Assay Kit (Colorimetric; Abcam ab83369), in accordance with product‐recommended protocol. The mESCs were grown and transfected in 48‐well TC plates. Optical densities at 405 nm were read with the Tecan Infinite M1000 PRO Multimode Microplate Reader in 96‐well clear flat bottom TC plates.
3
Rab7 GAP Activity Assay Using TBC1D5 and Retromer
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The Rab7 GAP activity of TBC1D5 in the presence of retromer and cyclic peptides was carried out using the GTPase-Glo assay kit (Promega) according to the manufacturer’s instructions. Briefly, 10 μM Rab7a containing 10 μM GTP and 1 mM DTT in GTPase/GAP buffer was incubated with 3 μM TBC1D5TBC, 6 μM retromer, and 60 μM cyclic peptides or equivalent percentage (v/v) of DMSO for 60 min at room temperature. For the Rab7a-only experiment, 10 μM Rab7a was incubated with 60 μM cyclic peptides without TBC1D5TBC or retromer. Similarily, TBC1D5TBC-only experiments were performed by incubating 10 μM Rab7a with 3 μM TBC1D5TBC and 60 μM cyclic peptides without retromer. Opposite reactions were also prepared for retromer only experiments. To complete the GTPase reaction, an equal volume of the reconstituted GTPase-Glo reagent containing 5 μM adenosine diphosphate was then added to the reaction mix and incubated for a further 30 min at room temperature. The luminescence of residual GTP after GTPase reaction was measured by adding detection reagent to the reaction mix and recorded using an Infinite M1000 PRO multimode microplate reader (Tecan) at 25°C.
4
Myc Reporter Cell-Based Assay
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The HCT116 Myc‐Luc reporter cells were reverse‐transfected in 384‐well plates. Transfection mix consisting of transfectants, Lipofectamine 2000 (used according to manufacturer's recommendations), and Opti‐MEM was pipetted into respective wells (20 μl per well). 5,500 cells in 30 μl of 16.7% FBS‐supplemented DMEM media were then seeded in each well. After 72 h, cell viability was assayed for using PrestoBlue reagent (Thermo Fisher Scientific A13262), followed by Steady‐Glo Luciferase Assay (Promega E2550) for Myc activity. Fluorescence and luminescence readings were obtained with the Tecan Infinite M1000 PRO Multimode Microplate Reader.
5
Determination of Total Phenolic Content
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The measurements of TPCs were performed in microplates (Infinite M1000 Pro Multimode Microplate Reader, Tecan, Männedorf, Switzerland) using the procedure of Horszwald and Andlauer [24 (link)]. The TPC assay was performed in microplates, and aliquots of 15 μL of methanol extracts were placed in microplate wells. Subsequently, 250 μL of the Folin–Ciocalteu reagent (previously diluted with water, 1:15, v/v) were added, and the mixture was incubated for 10 min in the dark at room temperature. Then, 25 μL of 20% sodium carbonate were added to each well. The absorbance of the mixture in the measurement of TPC was evaluated with the Folin–Ciocalteu reagent at 755 nm after 5 min of reaction in microplates. The results were calculated according to prepared standard curves of gallic acid in the range of 0.03–1.0 mg L−1 and presented as milligrams of gallic acid equivalent (GAE) per gram of sample (mg GAE g−1). Three replicates were analyzed for each honey type.
6
Quantifying Intracellular ROS in Hepatocytes
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Intracellular ROS in the primary hepatocytes were assessed using the fluorescence based CellRox™ (ThermoFischer Scientific) method. Fluorescence was recorded in the Infinite M1000 Pro Multimode microplate reader (Tecan). Dead cells were assessed by staining with SYTOX™ (ThermoFischer Scientific) according to the manufacturer's instructions and eliminated from the calculations of ROS levels.
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