The activity of caspase-1, -3, -4, -5, -6, -7, -8 and -9 was measured using a Muse® Multi-caspase kit (MCH100109, Merck Millipore, MA, USA) according to the manufacturer's instructions. MDA-MB-231 cells were incubated with various concentrations of ATN-RNA (0; 10; 25; 50 and 100 nM) for 24 h. Cells were harvested and incubated with 5 μl of Muse® Multi-Caspase reagent working solution at 37°C for 30 min. Then, 150 μl of Muse® Caspase 7-AAD working solution was added to each sample. Multi-caspase assay was performed with Muse® Cell Analyzer (Merck Millipore, MA, USA).
Muse multicaspase kit
Manufactured by Merck Group
Sourced in United States
The Muse™ MultiCaspase Kit is a flow cytometry-based assay that quantitatively measures the activity of multiple caspases, which are key enzymes involved in the apoptosis (programmed cell death) process. The kit provides a simple, rapid, and sensitive method for detecting and analyzing caspase activation in cell samples.
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Lab products found in correlation
Muse cell analyzer, by Merck Group (4 mentions)
Muse mitopotential kit, by Merck Group (3 mentions)
Muse cell cycle kit, by Merck Group (2 mentions)
Caspase colorimetric assay kit, by Abcam (2 mentions)
Annexin 5 alexa fluor 568 conjugate, by Thermo Fisher Scientific (2 mentions)
Cytoselect 48 well cell adhesion assay, by Cell Biolabs (1 mentions)
Primesurface96u multiwell plates, by Sumitomo Bakelite (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Facscalibur system, by BD (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (1 mentions)
Muse cell analyser, by Merck Group (1 mentions)
Caspase 12 fluorometric assay kit, by Abcam (1 mentions)
Muse oxidative stress kit, by Merck Group (1 mentions)
Caspase 9 fluorometric assay kit, by Abcam (1 mentions)
Muse mitopotential assay kit, by Merck Group (1 mentions)
Caspase 3 fluorometric assay kit, by Abcam (1 mentions)
21 protocols using muse multicaspase kit
1
Measuring Caspase Activity in MDA-MB-231 Cells
2
Multiparametric Caspase Activation Assay
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The activation of caspases was determined using the Muse™ MultiCaspase Kit (Merck®; cat. no.: MCH100109, Poznań, Poland). The whole procedure was performed accordingly to manufacturer's protocol and our previous experiment.35 Briefly, Caspase buffer was diluted 10× in DEPC‐treated water and the MultiCaspase Reagent Stock Solution was diluted in 50 µL of DMSO. Other reagents were prepared for the analysis as it was described elsewhere.35 According to manufacturer instructions, the analysis of caspases activation was based on membrane permeable VAD‐peptide that can detect multiple caspases for example caspase 1, −3, −4, −5, −6 −7, −8 and −9. Stained samples were incubated for 30 min (37°C, 95% humidity, 5% CO2) and 150 µL of MuseTM 7‐AAD Working Solution was added in order to detect dead cells. The caspases activity profile was measured using MuseTM Cell Analyzer. Each analysis was performed in triplicate. The gating procedure of cells’ populations was based on the positive and negative controls.32 , 33 , 34
3
Multiparametric Analysis of Caspase Activation
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Analysis of caspase activation was performed using the Muse™ MultiCaspase Kit (Merck®; cat. no.: MCH100109, Poznan, Poland). The assay was carried out in accordance with the protocol published by the manufacturer. Prior to the assay, cells were harvested as described above (Section 2.2 .). Caspase buffer supplied with the kit was diluted 10 times using diethylpyrocarbonate-treated water (DEPC-treated water). MultiCaspase Reagent Stock Solution was prepared by adding 50 μL DMSO to the included MultiCaspase Reagent dye. MultiCaspase Reagent Working Solution was prepared at the dilution of 1:160 in 1 × PBS. Caspase 7-AAD Working Solution was prepared by adding 2 μL of the Muse™ Caspase 7-AAD dye to 148 μL of 1 × Caspase Buffer. After trypsynization, 50 μL of cells were suspended in 5 μL of the MuseTM MultiCaspase Working Solution reagent. After 30 min of incubation in a CO2 incubator (37 °C, 95% humidity, 5% CO2) 150 μL MuseTM 7-AAD Working Solution was added. The activity profile of the caspases was determined using the MuseTM Cell Analyzer. Each analysis was performed in triplicate.
4
Cell Proliferation, Migration, Invasion, and Adhesion Assays
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We used a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) for cell proliferation, a wound-healing assay for cell migration, BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) for cell invasiveness, and the CytoSelect 48-Well Cell Adhesion Assay (Cell Biolabs, San Diego, CA, USA) for cell adhesion to extracellular matrix components. These assays were performed as previously described.2 (link),40 (link) To evaluate total caspase activity, a Muse MultiCaspase Kit (Merck Millipore, Billerica, MA, USA) was used. The activities of caspase-3, -8, -9, and -12 were measured using the Caspase Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). Mitochondrial membrane potential and cell-cycle distribution were assessed using a Muse MitoPotential Kit (Merck Millipore) and a Muse Cell Cycle Kit (Merck Millipore), respectively. ALDH, a surrogate marker of stem/progenitor cells, was estimated using the ALDEFLOUR fluorescent reagent system (STEMCELL Technologies, Vancouver, BC, Canada). ALDH-positive cells were determined using a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). The three-dimensional spheroid cultures were analyzed using PrimeSurface96U multiwell plates (Sumitomo Bakelite, Tokyo, Japan).
5
Multi-Caspase Activity Assay
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Multi-caspase (caspase-1, -3, -4, -5, -6, -7, -8, and -9) activity was analyzed on the basis of the cleavage of caspase substrate using a Muse™ Multi-Caspase kit from Merck Millipore. The analyses were conducted according to the protocol provided by the manufacturer. The cells were exposed to Muse™ Multi-Caspase Reagent working solution for 30 min at 37 °C and Muse™ Caspase 7-AAD working solution was added, followed by incubation for five min at RT in the dark. The stained samples were analyzed using a Muse™ Cell Analyzer (Merck Millipore).
6
Apoptosis Quantification in ETNK2 KO Cells
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Apoptotic cells were detected by staining with annexin V-Alexa Fluor 568 conjugate (A13202, Thermo Fisher Scientific).19 (link) Briefly, parental or stable ETNK2 KO GC cell lines (1 × 105 cells/ml) were mixed with 10 µl of annexin V conjugate and incubated for 15 min. Cells irradiated with ultraviolet light for 120 min served as a positive control. The cells were visualised by phase contrast and fluorescence microscopy using a BZ9000 microscope (Keyence, Osaka, Japan). To quantify the percentage of cell apoptosis, the total number of cells and annexin V-positive cells in eight randomly selected fields were counted. To measure mitochondrial transmembrane potential and total caspase activity, 5.0 × 104 cells/condition were collected and analysed using a Muse MitoPotential Kit or a Muse MultiCaspase Kit (Merck Millipore, Billerica, MA, USA), respectively, according to the manufacturer’s protocols.20 (link),21 (link)
7
Comprehensive Cell Assays for Proliferation, Apoptosis, and Cell Cycle
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Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Apoptosis was measured using an annexin V-Alexa Fluor 568 conjugate (A13202, Thermo Fisher Scientific). Total caspase activity was measured using a Muse Multi-caspase kit (MCH100109, Merck Millipore, Billerica, MA, USA). A Caspase Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) was used to measure the activity of caspase-3, 8, 9, and − 12 individually. The Muse MitoPotential kit (Merck Millipore) was used to assess the mitochondrial membrane potential. Cell cycle progression was analyzed using a Muse Cell Cycle Kit (Merck Millipore). We also analyzed the cell cycle using a Cell Cycle Assay Cell-Clock Kit (Biocolor, Carrickfergus, UK).
8
Detecting Caspase Activity in Cancer Cells
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The Muse™ MultiCaspase Kit (Merck Millipore, Billerica, MA, USA, Catalog No. MCH100109) was used to detect the activity of multiple caspases (caspase-1, 3, 4, 5, 6, 7, 8, 9) in cancer cells. Due to using a derivatized VAD-peptide and a dead cell dye, the assay allows for identification of the activity of caspases and distinguishes four population of cells: Live, Caspase (+), Caspase+/Dead and Dead. The results of the assay were measured using the Muse™ Cell Analyzer (Merck Millipore, Billerica, MA, USA).
9
Curcumin and Arsenic Trioxide Induce Caspase Activation
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The MCF-7 cells were cultured in 6-well plates, overnight; starved for 12 h and treated for 24 h with curcumin, which also served as a positive control and arsenic trioxide concentrations (11 and 32 μM). After treatment with the arsenic trioxide and the positive control (curcumin), the percentages of cells with activated caspases were quantified using the Muse™ Multi-Caspase kit following the manufacturer’s instructions (Merck Millipore) and analyzed using the Muse Cell Analyzer.
10
Apoptosis Induction by As2O3 Quantified
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To classify apoptosis induced by As2O3, the Multi-Caspase Assay was performed as instructed by the Muse® Multi-Caspase Kit protocol (Merck Millipore). MFC-7 cells (2 × 105 cells/well) were cultured in six well plates and allowed to adhere to the bottom of the plate, overnight. Following the incubation overnight, the cells were treated with 32 µM As2O3 and 100 µM curcumin for 24 h, in appropriate cell culture conditions. Following treatment, the cells were trypsinized and centrifuged at 300× g for 5 min. Multi-Caspase buffer was used to re-suspend the cells and 50 µL of each sample was transferred to a new tube and 5 µL of Multi-Caspase reagent was added. Samples were mixed and incubated at 37 °C in CO2 incubator for 30 min, followed by addition of 150 µL 7-AAD and placed in the dark for 5 min at room temperature. The samples were then analysed using the Muse® Cell Analyser (Merck-Millipore).
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