The measurement of oxidative stress cells was quantified by Muse Oxidative Stress Kit (MCH100111, Merck Millipore). The cells were seeded in 6-well plates overnight and then treated with DPT (0, 6, or 8 nM) or 1 μM GEF for 48 h. Cells were harvested and washed with assay buffer, and then incubated with Muse Oxidative Stress Reagent working solution in the dark at 37°C for 30 min. The percentage of cells undergoing oxidative stress were identified using the Muse Cell Analyzer and Muse analysis software (Merck Millipore).
Muse analysis software
Manufactured by Merck Group
Sourced in United States
Muse analysis software is a data analysis tool developed by Merck Group. The software is designed to streamline the processing and analysis of data generated from various laboratory instruments and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Muse cell analyzer, by Merck Group (15 mentions)
Muse oxidative stress kit, by Merck Group (4 mentions)
Muse cell cycle kit, by Merck Group (2 mentions)
Muse cell cycle reagent, by Merck Group (1 mentions)
Muse annexin 5 dead cell assay kit, by Merck Group (1 mentions)
Muse autophagy lc3 antibody based kit, by Merck Group (1 mentions)
Dead cell kit, by Merck Group (1 mentions)
Mts test, by Promega (1 mentions)
Muse bcl 2 activation dual detection kit, by Merck Group (1 mentions)
Muse cell cycle assay kit, by Merck Group (1 mentions)
Muse annexin 5 and dead cell kit, by Merck Group (1 mentions)
Muse annexin 5 dead cell kit, by Merck Group (1 mentions)
15 protocols using muse analysis software
1
Quantifying Oxidative Stress Response
2
Quantification of Cellular Oxidative Stress
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Determination of ROS was performed by using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA); 100 μM DCFH-DA, dissolved in 100% methanol which was added to the cellular medium and the cells were incubated at 37 °C for 30 min. Under these conditions, the acetate group is not hydrolyzed [54 (link)]. The fluorescence [corresponding to the oxidized radical species 2′,7′-dichlorofluorescein (DCF)] was monitored spectrofluorometrically (excitation, λ = 488 nm; emission, λ = 525 nm). The total protein content was evaluated for each sample, and the results were reported as percentage increase in fluorescence intensity (FI)/mg protein with respect to control untreated cells.
The quantitative measurement of cellular populations undergoing oxidative stress was performed using the Muse Oxidative Stress Kit (Merck Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. This assay utilizes dihydroethidium (DHE), which is cell membrane-permeable and, upon reaction with superoxide anions, undergoes oxidation to form DNA-binding fluorophore. The kit determines the percentage of cells that are negative [ROS(−)] and positive [ROS(+)] for reactive oxygen species. The count and percentage of cells undergoing oxidative stress were quantified using the Muse Cell Analyzer and Muse analysis software (Merck Millipore, Milano, Italy).
The quantitative measurement of cellular populations undergoing oxidative stress was performed using the Muse Oxidative Stress Kit (Merck Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. This assay utilizes dihydroethidium (DHE), which is cell membrane-permeable and, upon reaction with superoxide anions, undergoes oxidation to form DNA-binding fluorophore. The kit determines the percentage of cells that are negative [ROS(−)] and positive [ROS(+)] for reactive oxygen species. The count and percentage of cells undergoing oxidative stress were quantified using the Muse Cell Analyzer and Muse analysis software (Merck Millipore, Milano, Italy).
3
Cell Cycle Analysis of A549 and H460 Cells
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Cell cycle components were analyzed with Muse™ cell cycle reagent (EMD Millipore Corp [40 (link)]. Billerica, MA, USA). A549 and H460 cells (10 × 104 cells/well) were seeded in six-well plates and treated with CTT (0, 5, or 10 μM) or GF 20 μM for 16 h. After being harvested from the culture medium, cells were washed with 1mL PBS, then fixed with ice-cold fresh 70% EtOH for over 3 h. Next, cells were washed with PBS and had Muse™ cell cycle reagent added to them. Then, cells were incubated at room temperature for 30 min without light. Cells were analyzed with Muse cell analyzer and Muse analysis software (Merck Millipore).
4
Quantitative Measurement of Cellular Oxidative Stress
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The quantitative measurement of cellular populations undergoing oxidative stress was performed using the Muse Oxidative Stress Kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. This assay utilizes dihydroethidium (DHE), which is cell membrane-permeable and upon reaction with superoxide anions undergoes oxidation to form DNA-binding fluorophore. The kit determines the percentage of cells that are negative [ROS(−)] and positive [ROS(+)] for reactive oxygen species. Briefly, 1 × 106 cells/ml were harvested, washed with PBS, and then incubated in the dark at 37 °C for 30 min with the Muse Oxidative Stress Reagent working solution, which contained DHE. The count and percentage of cells undergoing oxidative stress were quantified using the Muse Cell Analyzer and Muse analysis software (Merck Millipore, USA).
5
Cell Cycle Analysis of HBx and UCP
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For confirming the cell cycle effect of HBx and UCP expression, the cells were transduced with Ad-F-UCP or Ad-LacZ at a 50 MOI for 24 h. Then, the cells were fixed with 70% ethanol, washed with ice-cold PBS, and the cells were incubated with a Muse cell cycle kit (#MCH100106, EMD Millipore, Hayward, CA, USA) at RT for 30 min and analyzed on a Muse™ cell analyzer (EMD Millipore). Data were quantified using the Muse™ analysis software (EMD Millipore).
6
Quantitative Measurement of Oxidative Stress
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The quantitative measurement of cellular populations undergoing oxidative stress was performed using the Muse Oxidative Stress Kit (Merck Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. This assay utilizes dihydroethidium (DHE), which is cell membrane-permeable and, upon reaction with superoxide anions, undergoes oxidation to form DNA-binding fluorophore. The kit determines the percentage of cells that are negative [ROS(−)] and positive [ROS(+)] for reactive oxygen species. Briefly, after 6 h of treatment, 1 × 106 cells/mL were harvested, washed with PBS, and then incubated in the dark at 37 °C for 30 min with the Muse Oxidative Stress Reagent working solution, which contained DHE. The count and percentage of cells undergoing oxidative stress were quantified using the Muse Cell Analyzer and Muse analysis software (Merck Millipore, USA).
7
Evaluating Autophagy-Induced Apoptosis in RCC Cells
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For confirming the apoptosis-inducing effect of the autophagy compound libraries on RCC cells, the cells seeded into a 24-well plate (5 × 104 cells/well) were treated with 5 µM autophagy compound libraries for 24 h. Then, the cells were harvested, washed with ice-cold PBS, and re-suspended in complete DMEM. The cells (1 × 104 cell/100 µl) were incubated with a Muse™ annexin V & dead cell assay kit (EMD Millipore, Hayward, CA, USA) at RT for 20 min and analyzed on a Muse™ cell analyzer (EMD Millipore). Data were quantified using the Muse™ analysis software (EMD Millipore). For autophagy detection in RCC cells after treatment with autophagy inducing compounds, the cells were treated with 5 µM autophagy compound libraries for 24 h. Subsequently, the cells were harvested, incubated with a Muse autophagy LC3-antibody based kit (EMD Millipore) according to the manufacturer’s protocol, and analyzed using a Muse™ cell analyzer.
8
Apoptosis Quantification in A549 and H460 Cells
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The extent of apoptosis was determined by Muse Annexin V and a dead cell kit (Millipore, Billerica, MA, USA). According to the manufacturer’s procedure, A549 and H460 cells (10 × 104 cells/well) were seeded in 6-well plates and treated with CTT (0, 5, or 10 μM) or GF 20 μM for 24 h. After being harvested from the culture medium, cells were washed with 1 mL PBS and 100 μL of Muse™ Annexin V Dead Cell reagent were added to them. Next, cells were incubated at room temperature for 20 min without light. The apoptosis was then analyzed with Muse cell analyzer and Muse analysis software (EMD Millipore).
9
Evaluation of X-PDT Cytotoxicity
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The in vitro toxicity of X-PDT was
evaluated using the MTS test. Before MTS treatment, HCT 116 cells
and CCD 841 CoN cells (2 × 104 mL–1) were grown on 96-well plates in the culture medium with 10% FBS
for 24 h. After removing the old media, the cells were incubated with
PLGA–TPP samples diluted in the culture medium for 4 h. After
incubation, the old medium was removed, and fresh medium was added,
followed by X-ray radiation at 4 Gy. The cytotoxicity of X-ray-induced
PDT on HCT 116 cells at 24 h was determined by the MTS test (Promega
Co., Fitchburg, WI) according to the manufacturer’s instructions
and compared with control cells without any treatment. Cell viability
was then calculated as a percentage of the absorbance of the untreated
control sample. For a comparison, the viability of cells treated with
PLGA–TPP alone and X-ray at 4 Gy alone was also evaluated in
the same experimental conditions. HCT116 cells (1 × 105 cells/well) were seeded in 25 cm2 cell culture flasks
and treated with X-ray alone, PLGA–TPP nanoparticles alone,
and X-PDT. At 24 h after treatments, the cells were harvested from
the culture medium and washed with 1 mL of PBS, followed by the addition
of 100 μL of Muse Annexin V Dead Cell reagent. Next, cells were
incubated at room temperature for 20 min without light. Apoptosis
was then analyzed with Muse Cell Analyzer and Muse analysis software
(EMD Millipore).
evaluated using the MTS test. Before MTS treatment, HCT 116 cells
and CCD 841 CoN cells (2 × 104 mL–1) were grown on 96-well plates in the culture medium with 10% FBS
for 24 h. After removing the old media, the cells were incubated with
PLGA–TPP samples diluted in the culture medium for 4 h. After
incubation, the old medium was removed, and fresh medium was added,
followed by X-ray radiation at 4 Gy. The cytotoxicity of X-ray-induced
PDT on HCT 116 cells at 24 h was determined by the MTS test (Promega
Co., Fitchburg, WI) according to the manufacturer’s instructions
and compared with control cells without any treatment. Cell viability
was then calculated as a percentage of the absorbance of the untreated
control sample. For a comparison, the viability of cells treated with
PLGA–TPP alone and X-ray at 4 Gy alone was also evaluated in
the same experimental conditions. HCT116 cells (1 × 105 cells/well) were seeded in 25 cm2 cell culture flasks
and treated with X-ray alone, PLGA–TPP nanoparticles alone,
and X-PDT. At 24 h after treatments, the cells were harvested from
the culture medium and washed with 1 mL of PBS, followed by the addition
of 100 μL of Muse Annexin V Dead Cell reagent. Next, cells were
incubated at room temperature for 20 min without light. Apoptosis
was then analyzed with Muse Cell Analyzer and Muse analysis software
(EMD Millipore).
10
Cell Cycle Analysis using Muse Kit
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Muse Cell Cycle Kit (Luminex, TX, USA) was used to analyze the cell cycle. HES-treated cells were trypsinized from the culture medium, centrifuged at 300 g for 5 min. Then phosphate-buffered saline (PBS) was used to wash the cells. 70% cold ethanol were used to xed the cells in at -20°C. The cells were then centrifuged and exposed to cell cycle reagents for 30 minutes without light. A Muse cell analyzer and Muse analysis software (Merck, Darmstadt, Germany) were used to analyze the data.
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