Glycogen content was determined as described in Gründel et al. (2012) (link) with minor modifications. A 2 ml aliquot of culture was harvested by centrifugation (15 000 g for 10 min at 4 °C) and stored at –20 °C after the supernatant was carefully removed. Frozen pellets were resuspended in 30% KOH, vigorously mixed by vortexing for 3 min, and incubated for 2 h at 95 °C. Cold ethanol was added for glycogen precipitation and incubated overnight at –20 °C. Glycogen was purified after two cycles of centrifugation (15 000 g for 10 min at 4 °C) and washed with cold ethanol, ensuring total elimination of KOH traces. After drying the pellet, it was resuspended in 100 mM sodium acetate (pH 5.2) and digested with 10 U of amyloglucosidase (form Aspergillus niger, Sigma) at 55 °C overnight in parallel with a calibration curve using glycogen (from bovine liver, Sigma). Glucose released in the process was quantified with a coupled enzyme reaction of glucose oxidase (from A. niger, Sigma) and peroxidase (from horseradish, Sigma) in the presence of O-dianisidine (Sigma) to a final concentration of 200 mg ml–1, 25 U ml–1, and 5 U ml–1, respectively, for 30 min at 30 °C. The reaction was stopped by adding H2SO4 to a final concentration of 4.8 N, and absorbance was registered at 540 nm on a Varioskan multiplate reader (Thermo Fisher Scientific).
Varioskan multiplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Varioskan multiplate reader is a versatile and efficient instrument designed for high-throughput analysis of biological samples. It is capable of performing various spectrophotometric and fluorometric measurements on microplates, allowing researchers to analyze a large number of samples simultaneously. The core function of the Varioskan multiplate reader is to provide accurate and reliable data for a wide range of applications in life science research and clinical diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
O dianisidine, by Merck Group (1 mentions)
Peroxidase from horseradish, by Merck Group (1 mentions)
Aspergillus niger, by Merck Group (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Hfobs, by American Type Culture Collection (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
6 protocols using varioskan multiplate reader
1
Glycogen Quantification in Microbial Cultures
2
Fluorimetric HDAC Activity Assay
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In-vitro histone deacetylase activity assay was performed using a fluorimetric HDAC activity assay kit (Sigma, catalog number CS1010) containing a fluorogenic HDAC substrate Boc-Lys(Ac)-AMC to examine the histone deacetylase activity of AtHDA7 HD. Since AtHDA7 belongs to the class I family of HDACs and the Boc-Lys(Ac)-AMC fluorogenic substrate is more specifically identified by class I HDACs, this substrate was used for the activity assay. The assay buffer was mixed with 81 μM, 162 μM and 324 μM of AtHDA7 HD separately, followed by mixing with 50 μl of 200 mM fluorogenic HDAC substrate solution in a clear, flat bottom, black 96-well assay plate. Similarly, AtHDA18 HD as a positive control, at concentrations of 81 μM, 162 μM, and 324 μM, was mixed with assay buffer and substrate. Three separate reactions for substrate solution mixed with assay buffer were used as negative control. The reactions were then incubated for 30 min at 30 °C, followed by adding 10 μl developer solution and further incubated for 20 min at room temperature. Finally, the fluorescence intensity of reactions was measured using a Varioskan multi-plate reader (Thermo Fisher) at an excitation wavelength of 350 nm and an emission wavelength of 460 nm. Each reaction was performed in triplicate.
3
NF-κB Luciferase Assay in MEFs
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Luciferase activity of NF-κB reporter was determined luminometrically using the Dual-Glo luciferase assay system (Promega). MEFs (106) were transiently transfected with pGL4.74-hRluc/TK (10 ng) and pGL4.10.NF-κB reporter (100 ng) vectors along with the indicated constructs. Luciferase activity of cell extracts in passive lysis buffer was determined luminometrically using the Varioskan multiplate reader (Thermo Fisher Scientific).
4
Flg22-Induced ROS Assay in Leaves
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ROS assays were performed as described previously [41 (link)]. Briefly, 16 leaf discs were excised per genotype of four weeks-old plants and treated with 1 μM flg22. ROS was measured with a Varioskan multiplate reader (Thermo Fisher Scientific, USA) for 35 min.
5
Evaluating Cell Viability via MTT Assay
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The influence of the scaffolds on cell viability was evaluated via the reduction reaction of the tetrazolium salt MTT (3(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), purchased from Sigma Aldrich, Germany. Scaffolds were exposed to the UV light for 30 min for sterilization. The samples were soaked into 3 mL of Advanced DMEM supplemented with 5% FBS and incubated for 24 h at 37°C in an atmosphere containing 5% CO2. The human bone tissue derived osteoblast cells (hFOBs, ATCC, UK) (10,000 cells/well) were seeded into a 96-well microtitre plate with a final volume of 100 μL of Advanced DMEM with 5% FBS. The material samples (supernatants of the starting samples) were added to the cells after 24 h of incubation at 37°C in 5 wt.% CO2 in four parallels and in several dilutions (undiluted, 1:2, 1:4, 1:8 and 1:16). As control, Advanced ADMEM and 5% FBS was added to the cells. After 24 h of treatment, cell viability was determined using the MTT test (Maver et al., 2018 (link); Stergar et al., 2017 (link)). For this purpose, 10 wt.% reagent was added to the medium and discarded after 4 h. Then, 100 μL of DMSO was added and after 5 min, the absorbance was measured spectrophotometrically at 570 nm using the Varioskan multiplate reader (ThermoFisher, Germany).
6
ROS Quantification in IC50-treated Cells
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ROS production in the IC50-treatment-challenged cells was quantified using the 2′,7′-dichlorodihydrofluorescein acetate reagent (H2DCFDA, Invitrogen, Waltham, MA, USA). Briefly, the cells (HT29 and HCT116, 1 × 104 cells/well) were seeded in black 96-well plates for 24 h. After the incubation, the cells were treated with the IC50 concentrations of CIE, RIE, BITC, and AITC for 24 h. Then, 5 µM H2DCFDA was added, and the cells were incubated for 45 min in the dark. The fluorescence was measured at 480/530 nm excitation/emission intensities in a Varioskan multiplate reader (ThermoScientific). The results were expressed as relative fluorescence units (RFU).
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