Proapoptotic effects of single compounds and their combinations were analyzed by the measurement of mitochondrial membrane potential (ΔΨm) using the Muse® MitoPotential Kit (Merck, Darmstadt, Germany) according to the manufacturer’s recommendations. Briefly, 2 × 105 cells per well were seeded in 6-well plates and preincubated for 24 h. Then, the appropriate growth medium was replaced with fresh medium (2 mL per well) containing IC25 concentrations of the tested glycolysis inhibitors or their combinations with Wnt signaling inhibitors, and the cells were incubated for an additional 48 h. The cells incubated with solvent served as a negative control. Additionally, cells incubated with 10 µM CCCP (carbonyl cyanide 3-chlorophenylhydrazone; Sigma-Aldrich, St. Louis, MI, USA) served as a positive control of mitochondrial membrane depolarization. Afterwards, the cells were collected by trypsinization, resuspended in 100 µL of assay buffer, 100 µL of MitoPotential working solution and incubated for 20 min at 37 °C in a humidified CO2 incubator. Fluorescence was analyzed by flow cytometry on a Muse® Cell Analyzer (Merck, Darmstadt, Germany). Data were evaluated using Muse® 1.5 analysis software (Merck, Darmstadt, Germany). All the experiments were done in triplicate.
Muse 1.5 analysis software
Manufactured by Merck Group
Sourced in Germany, United States
Muse 1.5 Analysis Software is a data analysis tool developed by Merck Group. It is designed to process and visualize data from various laboratory instruments. The software provides a comprehensive suite of analytical capabilities to support researchers in interpreting their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Muse cell analyzer, by Merck Group (14 mentions)
Topotecan, by Merck Group (5 mentions)
Muse cell cycle kit, by Merck Group (4 mentions)
Muse cell analyzer system, by Merck Group (2 mentions)
Muse mitopotential kit, by Merck Group (1 mentions)
Muse cell cycle reagent, by Merck Group (1 mentions)
Muse annexin 5, by Merck Group (1 mentions)
Dead cell reagent, by Merck Group (1 mentions)
Muse annexin 5 and dead cell kit, by Merck Group (1 mentions)
Muse oxidative stress kit, by Merck Group (1 mentions)
Caspase 3 7 reagent, by Merck Group (1 mentions)
Caspase 7 aad, by Merck Group (1 mentions)
Muse ki67 proliferation kit, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Muse annexin 5 dead cell reagent, by Merck Group (1 mentions)
Muse caspase 3 7 kit, by Merck Group (1 mentions)
16 protocols using muse 1.5 analysis software
1
Mitochondrial Potential Assessment of Glycolysis and Wnt Inhibitors
2
Cell Cycle Analysis by Flow Cytometry
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The analysis of the cell cycle distribution was performed using the Muse Cell Cycle Kit (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, cells (3 × 105 per well) were seeded and grown on 6-well plates for 24 h before stimulation. Then, the tested compounds were added and cells were incubated for 24 h. Topotecan (100 nM)-treated cells were used as a positive control for the cell cycle arrest. Subsequently, cells were harvested by trypsinization, washed with PBS buffer, fixed in ice-cold 70% ethanol, and frozen until staining at −20 °C. After overnight storage, cells were washed with cold PBS buffer, stained with propidium iodide in the presence of RNAase A, and, after 30 min incubation at room temperature and in the dark, the fluorescence signal was analyzed by flow cytometry on the Muse Cell Analyzer (Merck, Darmstadt, Germany). Data were evaluated using Muse 1.5 analysis software (Merck, Darmstadt, Germany).
3
Apoptosis Assay with Nafamostat and Palbociclib
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Cells were seeded in 12-well plates overnight, treated with various concentrations of nafamostat mesylate or combined with palbociclib for 96 h, and harvested after removing the medium. The harvested cells were incubated with MuseTM Annexin-V & Dead Cell kit reagent (Merck Millipore, Billerica, MA, USA) at 25 °C for 20 min in the dark. The apoptotic cells were measured using a MuseTM Cell Analyzer (Merck Millipore, Billerica, MA, USA), and data were analyzed using MUSE 1.5 Analysis software (Merck Millipore, Billerica, MA, USA).
4
Cell Cycle Analysis of PRI-724 and Inhibitor Combinations
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The effect of single compounds and combinations of PRI-724 with other inhibitors on cell cycle distribution was analyzed using the Muse® Cell Cycle Kit (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, 2 × 105 cells per well were seeded in 6-well plates. After 24 h of pre-incubation, the growth medium was replaced with fresh medium (2 mL per well) containing IC25 and/or IC50 concentrations of the compounds, and the cells were incubated for an additional 48 h. The cells incubated with DMSO served as a negative control, while cells incubated with 100 nM topotecan (Sigma-Aldrich, St. Louis, MI, USA) served as a positive control for cell cycle arrest. After incubation, cells were collected by trypsinization, washed with PBS buffer, fixed in ice-cold 70% ethanol, and stored overnight at −20 °C. The next day, fixed cells were collected by centrifugation and washed with PBS buffer. The distribution of cells depending on cell cycle phase (G1/G0, S, G2/M) was analyzed with the Muse® Cell Analyzer (Merck, Darmstadt, Germany) after staining with propidium iodide solution in the presence of RNase A for 30 min. The data were analyzed using Muse® 1.5 Analysis software (Merck, Darmstadt, Germany). All the experiments were done in triplicate.
5
Apoptosis Quantification by Flow Cytometry
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Apoptosis was assessed by the detection of phosphatidylserine externalization based on Annexin V staining combined with fluorescent DNA intercalator 7-Aminoactinomycin D (7-AAD) what allowed for the discrimination between early and late apoptotic cells. The analysis was performed by flow cytometry using the Muse Annexin V & Dead Cell Kit (Luminex, USA) and the Muse Cell Analyzer (Merck, Germany), according to the manufacturer’s protocol. Cells were seeded (2 × 105 cells per well) in six-well plates and on the following day the analyzed compounds were added. Topotecan (Sigma-Aldrich, USA) was used as a positive control of apoptosis induction. After 48 h cells were collected by trypsinization, stained and after 20 min incubation at room temperature in the dark analyzed by flow cytometry. Three independent experiments were performed. Data were analyzed by Muse 1.5 Analysis Software (Merck, Germany).
6
Mitochondrial Membrane Potential Assay
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Cells were seeded in 12-well plates overnight, treated with various concentrations of nafamostat mesylate or combined with palbociclib for 72 h, and harvested after removing the medium. The harvested cells were resuspended in Muse™ MitoPotential Kit reagent (Merck Millipore, Billerica, MA, USA), and cells were incubated at 37 °C for 20 min. 7-AAD was added, and cells were incubated at 25 °C for 5 min. The mitochondrial membrane potential of cells was determined using the Muse™ Cell Analyzer (Merck Millipore, Billerica, MA, USA), and data were analyzed using MUSE 1.5 Analysis software (Merck Millipore, Billerica, MA, USA).
7
Cell Cycle and Apoptosis Analysis
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Flow-cytometry analyses were performed using the Muse Cell Analyzer system and Muse 1.5 Analysis software (Merck, Darmstadt, Germany). For cell cycle analysis, the cells collected were first fixed in 70% ethanol for 12 hours, diluted to 3 × 105 cells/ml with Muse Cell Cycle Reagent (Merck) and incubated in the dark for 30 minutes prior to analysis. The number of events for analysis was set at 5000.
The cell cycle analyses were repeated but following a slightly different protocol whereby the media for cells that have been treated with BLE and GA at IC20 and IC50 for 48 hours, were replaced with fresh MEM, followed by a further 48-hours incubation (37 °C and 5% CO2)26 (link). Flow cytometry analyses were then performed as above.
The results for cell cycle analysis were expressed as DNA content profiles. For apoptosis analysis, Caco-2 cells were collected and resuspended in complete MEM at a concentration of 6 × 105 cells/ml. Two hundred microlitres of cell suspension was mixed with 200 μl of Muse Annexin V and Dead Cell Reagent (Merck) and incubated in the dark for 20 minutes prior to analysis. The number of events for analysis was set at 5000. The results for apoptosis analysis were expressed as apoptotic profiles.
The cell cycle analyses were repeated but following a slightly different protocol whereby the media for cells that have been treated with BLE and GA at IC20 and IC50 for 48 hours, were replaced with fresh MEM, followed by a further 48-hours incubation (37 °C and 5% CO2)26 (link). Flow cytometry analyses were then performed as above.
The results for cell cycle analysis were expressed as DNA content profiles. For apoptosis analysis, Caco-2 cells were collected and resuspended in complete MEM at a concentration of 6 × 105 cells/ml. Two hundred microlitres of cell suspension was mixed with 200 μl of Muse Annexin V and Dead Cell Reagent (Merck) and incubated in the dark for 20 minutes prior to analysis. The number of events for analysis was set at 5000. The results for apoptosis analysis were expressed as apoptotic profiles.
8
Annexin V-7-AAD Apoptosis Assay
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The externalization of phosphatidylserine was applied as a marker of apoptotic cells and analyzed using the Muse® Annexin V and Dead Cell Kit (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. The 7-aminoactinomycin D (7-AAD) stain was applied as a counterstain to Annexin V to discriminate between early and late apoptotic cells. Briefly, 2 × 105 cells per well were seeded in 6-well plates. After 24 h of pre-incubation, the growth medium was replaced with fresh medium (2 mL per well) containing IC25 and/or IC50 concentrations of the compounds, and the cells were incubated for an additional 48 h. The cells incubated with DMSO served as a negative control, while cells incubated with 100 nM topotecan (Sigma-Aldrich, St. Louis, MI, USA) served as a positive control for active apoptosis. Subsequently, the cells were collected and stained with Annexin V and 7-AAD in a culture medium solution. After 20 min of incubation, the cells were analyzed by flow cytometry on the Muse® Cell Analyzer (Merck, Darmstadt, Germany), and the data were further evaluated using Muse® 1.5 Analysis software (Merck, Darmstadt, Germany).
9
Flow Cytometry Analysis of AME Cytotoxicity
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For flow cytometry analyses, cells were exposed to AME concentrations of 0.5, 2.5, 5, 25, and 50 μM based on the previous results obtained in MTT assay. Muse Cell Analyzer system and Muse 1.5 Analysis software (Merck, Darmstadt, Germany) were used for performing flow cytometry analyses using Muse kits for cell cycle, apoptosis, and cell signaling in apoptosis (Annexin and Dead Cell kit, Cell Cycle Analysis kit, Caspase 3/7 kit, Bcl-2 Activation Dual Detection kit) according to the manufacturer’s instructions, and as already described in our previous studies [54 (link)]. The results are expressed as percentages of cells and represent the mean of three independent experiments. Doses lower than IC50 were used in the experiment assessing the AME effect on oxidative stress.
10
Quantifying Superoxide Radical Positive Cells
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Dihydroethidium (DHE) reacts with superoxide radicals, generating fluorophores with a high affinity to DNA. The Muse Oxidative Stress Kit (Merck, Darmstadt, Germany) containing DHE was used according to the manufacturer’s protocol for the quantitative measurement of superoxide radical positive cells. Briefly, cells (3 × 105 per well) were seeded in the 6-well plates. After 24 h, the tested compounds were added, and cells were incubated for 24 h. Subsequently, cells were collected by trypsinization, washed with PBS buffer, resuspended in 1X Assay Buffer, mixed with Muse Oxidative Stress Reagent working solution, and subjected to a 30 min incubation (37 °C, 95% humidity, 5% CO2). Cells were analyzed by flow cytometry on the Muse Cell Analyzer (Merck, Darmstadt, Germany), and data were evaluated using the Muse 1.5 Analysis Software (Merck, Darmstadt, Germany).
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