The cells (1 × 104 cells/well of a 96-well plate) were incubated with compound solutions (10 µM) during 1 h. Then, 6-OHDA/PQ/rotenone were added to cell suspension to resulting concentration of 50 µM, 500 µM and 10 µM respectively for incubation during 1 h. Cells incubated without neurotoxins and compounds and with neurotoxins alone were used as positive and negative controls, respectively. The 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (H2DCFDA, Molecular Probes, Eugene, OR, USA) was added to each well (10 µM, final concentration) and the plate was incubated for an additional 10 min at 37 °C. The intensity of dichlorofluorescein fluorescence was measured with PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany) at λex = 485 nm and λem = 518 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Germany). The results were presented as the percent of positive control data [71 (link)].
Mars data analysis v 3.01r2
Manufactured by BMG Labtech
Sourced in Germany
MARS Data Analysis v. 3.01R2 is a software package developed by BMG LABTECH for data analysis. The core function of this software is to provide users with tools for the processing and analysis of data generated from BMG LABTECH's microplate readers.
Automatically generated - may contain errors
Lab products found in correlation
Pherastar fs, by BMG Labtech (15 mentions)
Pherastar fs plate reader, by BMG Labtech (10 mentions)
H2dcfda, by Thermo Fisher Scientific (8 mentions)
2 7 dichlorodihydrofluorescein diacetate solution, by Thermo Fisher Scientific (2 mentions)
Daf fm fluorescent probe solution, by Thermo Fisher Scientific (2 mentions)
Sensolyte 520 sortase a activity assay kit, by AnaSpec (2 mentions)
6 ohda, by Merck Group (1 mentions)
Ascorbic acid, by Lumiprobe (1 mentions)
Sulfo cyanine5 azide, by Lumiprobe (1 mentions)
Daf fm, by Thermo Fisher Scientific (1 mentions)
Daf fm da, by Merck Group (1 mentions)
Sensolyte 520 sortase a activity assay kit fluorimetric, by AnaSpec (1 mentions)
A438079, by Merck Group (1 mentions)
Fluo 8 am, by Abcam (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Lumitrace cfda se kit, by Lumiprobe (1 mentions)
26 protocols using mars data analysis v 3.01r2
1
Neuroprotective Compound Evaluation in Cell-Based Assay
2
Neuroprotective effects of peptides against oxidative stress
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Neuro-2a cells were incubated with peptides at concentrations of 0.01, 0.1, and 1 µM for 1 h. Then, paraquat (600 μM) or rotenone (10 µM) was added to each well and cells were incubated for 3 or 1 h, respectively. To study the ROS formation, the 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay was performed according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA). H2DCF-DA solution was added to each well, such that the final concentration was 10 µM, then the microplate was incubated for an additional 30 min at 37 °C.
To determine the NO production, a 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) assay was performed according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). DAF-FM fluorescent probe solution was added to each well to a final concentration 5 μM, then the microplate was further incubated for 40 min at 37 °C. In both cases, the fluorescence intensity was measured using a PHERAstar FS high-speed plate reader (BMG Labtech, Ortenberg, Germany) at λex = 485 nm and λem = 518 nm. The data were processed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results are presented as percentages of control with inductor data.
To determine the NO production, a 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) assay was performed according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). DAF-FM fluorescent probe solution was added to each well to a final concentration 5 μM, then the microplate was further incubated for 40 min at 37 °C. In both cases, the fluorescence intensity was measured using a PHERAstar FS high-speed plate reader (BMG Labtech, Ortenberg, Germany) at λex = 485 nm and λem = 518 nm. The data were processed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results are presented as percentages of control with inductor data.
3
Assessing Neuroprotective Compounds against Oxidative Stress
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Cell suspensions (1 × 103 cells/well) were incubated with compound solutions (10 µM) for 1 h. Then, 6-OHDA at a concentration of 50 µM was added in each well, and cells were incubated for 30 min. In other experiments, cells were incubated with PQ at a concentration of 500 µM for 30 min and 1 h. Cells incubated without 6-OHDA/PQ and compounds, and with 6-OHDA/PQ only, were used as positive and negative controls, respectively. To study ROS formation, 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (Molecular Probes, Eugene, OR, USA) was added to each well, such that the final concentration was 10 mM, and the microplate was incubated for an additional 10 min at 37 °C. The intensity of dichlorofluorescein fluorescence was measured with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany) at λex = 485 nm, and λem = 518 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as a percentage of positive control data.
4
Rotenone-Induced ROS Assay in Neuro-2a Cells
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After 24 h of adhesion, Neuro-2a cells (1 × 104 cells/well) were incubated with compounds at concentrations of 0.1–10 µM for 1 h. Then, rotenone at a concentration of 10 µM was added in each well, and cells were incubated for 1 h. To study ROS formation, 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (H2DCF-DA, Molecular Probes, Eugene, OR, USA) was added to each well, such that the final concentration was 10 µM, and the microplate was incubated for an additional 30 min at 37 °C. The fluorescence intensity was measured using a high-speed plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany) at λex = 485 nm and λem = 518 nm. The results were processed in MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as percentages of positive control data.
5
Measuring Intracellular Oxidative Stress
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Cell suspensions (1 × 103 cells/well) were incubated during 1 h with 1 and 10 µM solutions of the tested compounds. Non-treated and treated with 6-OHDA at concentration of 50 µM (Sigma-Aldrich, USA) cells were used as negative and positive controls, respectively. The portion (20 µL) of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) stock solution (Molecular Probes, Eugene, OR, USA) with concentration of 100 mM was added in each well and the microplate was incubated for an additional 10 min at 37 °C. The intensity of dichlorofluorescin fluorescence was measured at λex = 485 nm, and λem = 518 nm with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany). The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). In other experiments, cells were incubated with compounds during 1 h. Then, 6-OHDA (50 µM) was added in each well for 30 min and ROS levels were measured. All obtained results were presented as percent of negative control data.
6
Quantifying Cellular Proliferation with EdU Assay
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The MCF-7 cells were seeded in a 96-well plate and treated with the compound at a concentration of 10 µM for 48 h. After 46 h of incubation, the dH2O solution of 5-ethynyl-2′-deoxyuridine (EdU) (Lumiprobe, Moscow, Russia) at a concentration of 10 µM was added to each well for 2 h, and then they were stained in accordance with the instructions of the manufacturer [54 ]. The cell monolayer was rinsed with phosphate-buffered saline (PBS) three times and permeabilized with 0.2% Triton X-100 (Helicon, Moscow, Russia) in PBS for 1 h at room temperature. Then, the cells were stained through a click reaction with ascorbic acid at 10 mM (Lumiprobe, Moscow, Russia), Cu(II)-TBTA complex at 2 mM (Lumiprobe, Moscow, Russia), and sulfo-Cyanine5 Azide at 8 µM (Lumiprobe, Moscow, Russia) in 100 mM Tris buffer pH 7.4 for 30 min at room temperature in the dark. After that, the cells were washed with PBS twice.
The fluorescence intensity was measured at λex = 485 and λem = 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany). The data were processed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany). The data are presented in relative fluorescent units.
The fluorescence intensity was measured at λex = 485 and λem = 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany). The data were processed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany). The data are presented in relative fluorescent units.
7
Quantifying Oxidative Stress Response in Cells
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The cells (1 × 104 cells/well of a 96-well plate) were incubated with compound solutions (1 and 10 µM) during 1 h. Then, PQ was added to cell suspension to a resulting concentration of 500 µM for incubation during 1 h. Cells incubated without PQ and compounds and with PQ alone were used as positive and negative controls, respectively. The 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (H2DCFDA, Molecular Probes, Eugene, OR, USA) was added to each well (10 µM, final concentration) and the plate was incubated for an additional 10 min at 37 °C. The intensity of dichlorofluorescin fluorescence was measured with a PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany) at λex = 485 nm and λem = 518 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as percent of positive control data.
8
Enzymatic Activity Assay for Sortase A
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The enzymatic activity of sortase A from Staphylococcus aureus was determined using SensoLyte 520 Sortase A Activity Assay Kit * Fluorimetric * (AnaSpec AS-72229, AnaSpec, San Jose, CA, USA) in accordance with the manufacturer’s instructions. DMSO at a concentration of 0.8% was used as a control. Fluorescence was measured with the plate reader PHERAStar FS (BMG Labtech, Offenburg, Germany) for 60 min with a time interval of 5 min. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Offen-burg, Germany). The results were presented as relative fluorescent units (RFUs) and percentage of the control data [28 (link)].
9
Evaluating Mitochondrial Dysfunction in Cells
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After 24 h of adhesion, the cells (1 × 104 cells/well of a 96-well plate) were incubated with different concentrations of 1,4-NQs (0.01–1.0 μM) for 1 h. Then, PQs or 6-OHDAs were added to cell to for incubation during 1 h or 5 h, respectively. The cells incubated without PQ or 6-OHDA and compounds and with PQ or 6-OHDA alone were used as positive and negative controls, respectively. The tetramethylrhodamine methyl (TMRM) (Sigma-Aldrich, St. Louis, MO, USA) solution at 500 nM was added in each well, and the cells were incubated for 30 min at 37 °C. The intensity of fluorescence was measured with a PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany) at λex = 540 nm and λem = 590 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as a percentage of positive control data [32 (link)].
10
Mitochondrial Membrane Potential Assay
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Neuro-2a cells (1.0 × 104 cells/well in a 96-well plate) were treated with different concentrations of compounds U-443 or U-573 (0.01–1.0 μM) for 1 h at 37 °C in a 5% CO2 incubator. Following this, a solution of rotenone at a concentration of 10.0 μM was added, and the cells were further incubated for 1 h. Subsequently, a solution of tetramethylrhodamine methyl (TMRM) at a concentration of 500 nM (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 30 min at 37 °C. Fluorescence intensity was measured using a PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany) with excitation at 540 nm and emission at 590 nm. Data analysis was performed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany), and the results were presented as a percentage of the control [55 (link)].
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