Silica gel (63–200 mesh; Merck, Germany) and Sephadex® LH-20 (25–100 μm) were the resins used for column chromatography (CC). The Silica gel 60 F-254 (Merck, Darmstadt, Germany) resin was used for thin-layer chromatography (TLC), with the compounds visualized by spraying with 10% (v/v) H2SO4 in an ethanol solution and heating for 10 min at 95 °C. HPLC chromatograms were obtained using an LC-6A instrument and an IOTA-2 RI-detector (Shimadzu, Kyoto, Japan). A Phenomenex Luna silica column (5 μm, 10 × 250 mm, 3 mL·min−1 flow rate) was used for normal-phase separations. To elucidate the chemical structures, the isolated active compounds were identified by nuclear magnetic resonance (NMR). 1H NMR spectra were run on a Bruker Avance-500 MHz FT NMR (Bruker, Rheinstetten, Germany) with CDCl3 and tetramethylsilane (TMS) as the solvent and internal standard, respectively. A multiscan microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used for the water-soluble tetrazolium salt (WST)-1 and NO assays.
Multiscan microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in Finland
The Multiscan microplate reader is a versatile instrument designed for absorbance measurements in microplates. It is capable of performing a wide range of absorbance-based assays, including colorimetric, fluorometric, and luminescent measurements. The device can accommodate various microplate formats and is suitable for a variety of applications in life science research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Silica gel 60 f254, by Merck Group (1 mentions)
Luna silica column, by Phenomenex (1 mentions)
Silica gel, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Keygen Biotech (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
7 protocols using multiscan microplate reader
1
Isolation and Structural Elucidation of Bioactive Compounds
2
Pro-ApoA-I ELISA Quantification in HepG2
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pro-ApoA-I protein concentrations in culture medium of HepG2 cells were measured by an enzyme-linked immunoassay (ELISA). The pro-ApoA-I ELISA was performed as described [8 (link)]. For the pro-ApoA-I ELISA measurements, the absorbances were determined at 450 nm using a multi-scan microplate reader (Thermo Fisher Scientific, Bleiswijk, Netherlands). Values are presented as relative fold changes compared to the control conditions.
3
Cell Viability Assay with CCK-8
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The cell viability was determined by the CCK-8 assay. Briefly, H9c2 cells were seeded in 96-well plates at a density of 1×105/ml, and 100 µl DMEM with 10% FBS was added to each well followed by incubation overnight. Following drug treatment, CCK-8 (10 µl/well) was added, and the cells were cultured for a further 2 h at 37°C. The absorbance at 450 nm was measured using a Multiscan microplate reader (Thermo Fisher Scientific, Inc.). The cell viability for the control group was set at 100%, while the viability for the other groups was expressed as a percentage of the control group.
4
Cytotoxicity Evaluation of 17BIPHE2 and RI-10
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Exponentially proliferating A549, NCI-H1975 and BEAS-2B cells were harvested from 96-well plates, and 5×103 cells/well were seeded in 96-well plates and cultured overnight at 37°C. The cells were treated with 0, 5, 10, 15, 20, 25, 30, 35 and 40 µmol/l 17BIPHE2 and RI-10 for 24 h (treatment group) respectively. 17BIPHE2 (1:10 solved in ddH2O) was not added to the control group, and no cells were added in the blank group. In total, four duplicate wells were used per group. After culturing for 24 h, the medium in the 96-well plate was removed; then, 50 µl MTT (5 g/l; Nanjing KeyGen Biotech Co., Ltd.) solution was added, and the cells were incubated in a cell culture incubator at 37°C. After 4 h, 150 µl dimethyl sulfoxide (DMSO) was added to each well and the plate was left to stand at room temperature. After reaction for 10 min, the absorbance at 490 nm was measured using a multiscan microplate reader (Thermo Fisher Scientific, Inc.). Percentage cell viability was calculated as follows: Cell viability %=[(A treatment-blank)/(A control-A blank)] ×100%, where A is the absorbance. The experiment was conducted in triplicate.
5
Colorimetric MTT Assay for Cytotoxicity
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A colorimetric assay using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was performed as was originally described by Mosmann, 1983 [69 (link)]. HCT-116 and HBL-100 cells were seeded in 96-well microplates (3–5 × 103 cells/well/100 μL, respectively). In both cell lines, 24 h later, the medium was aspirated and replaced by the medium containing treatments. B. grisebachii BLD was used at concentrations ranging from 0 to 2.000 μg/mL; while, the chemotherapic 5-fluorouracil (Filaxis®, Buenos Aires, Argentina) was used as a control cytotoxic compound at concentrations ranging from 0 to 125 μg/mL.
After 72 h of treatment, the medium was replaced by 100 μL of MTT solution (0.5 mg/mL in DMEM, without phenol red or FBS); and cells were incubated for an additional 4 h. MTT solution was then removed and 100 μL of DMSO added; the plates were shaken for 10 min to dissolve the formazan crystals. The optical density was measured using a Thermo Scientific Multiscan microplate reader at 570 nm. The optical density obtained in untreated control cells was taken as 100% viability. Assays were performed three times in triplicate.
After 72 h of treatment, the medium was replaced by 100 μL of MTT solution (0.5 mg/mL in DMEM, without phenol red or FBS); and cells were incubated for an additional 4 h. MTT solution was then removed and 100 μL of DMSO added; the plates were shaken for 10 min to dissolve the formazan crystals. The optical density was measured using a Thermo Scientific Multiscan microplate reader at 570 nm. The optical density obtained in untreated control cells was taken as 100% viability. Assays were performed three times in triplicate.
6
Huh7 and HepG2 Cell Proliferation
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Huh7 and HepG2 cells (4×10 4 ) were cultured with rIl-16 for 4, 16, and 24 h in 96-well flat bottom plates (Costar, NY, USA) in 200 L of RPM1-1640 medium. After incubation, 20
substrate was added to each well. Cell proliferation was evaluated using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) and the procedure was performed according to the manufacturer's instructions. Absorbance was measured at 490 nm with a Multiscan microplate reader (Thermo Labsystems, Vantaa, Finland). The proliferation rate was determined as the ratio of the optical density of treated to control cells.
substrate was added to each well. Cell proliferation was evaluated using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) and the procedure was performed according to the manufacturer's instructions. Absorbance was measured at 490 nm with a Multiscan microplate reader (Thermo Labsystems, Vantaa, Finland). The proliferation rate was determined as the ratio of the optical density of treated to control cells.
7
Evaluating Intracellular ROS in Leukemic Cells
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Initially, the screening of IP and leukemia cells was performed based on the redox effect. Then, the leukemic strain most responsive to treatments with the compounds (best oxidative profile) was selected for the following stages of the study. Intracellular ROS content was evaluated as reported by [53 (link)]. Human leukemic cell lines were incubated for 12 h with IP (10 μM), washed twice with HBSS, and then 100 μL of HBSS/well was added. After that, the cells were loaded with DCFH-DA (10 μM) in HBSS at 37 °C and incubated for 30 min. Excess DCFH-DA was removed by washing with fresh HBSS. The intensity of fluorescence was measured at 485 nm for excitation and 530 nm for emission using a Multiscan microplate reader (Thermo Fisher Scientific Oy®, Vantaa, Finland). The results of the fluorescence intensity of the compounds were obtained by discounting the baseline values of the cells not exposed to the compounds (negative control). A positive control with 100 µM H2O2 was performed.
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