Caspase-3/7 activity was quantified by using the Muse Caspase-3/7 assay kit (Merck Millipore Burlington, MA, USA) according to the manufacturer’s protocol. In short, U-118 MG cells grown in 24-well plates were treated with increasing concentrations of lycopene, [6]-gingerol, silymarin and DMSO for 24 and 48 h. Adherent cells were washed, collected and incubated with the MuseTM Assay Buffer BA, then incubated with MuseTM Caspase-3/7 Reagent, a hallmark of apoptosis for 30 min in the 37 °C incubator with 5% CO2 (Panasonic, Gunma, Japan). Afterwards, cells were incubated with MuseTM Caspase 7-AAD, a dead cell marker for 5 min at room temperature. The percentages of live (Caspase-3/7 (−) and 7-AAD (−)), dead (Caspase-3/7 (−) and 7-AAD (+)), apoptotic cells exhibiting Caspase-3/7 activity (Caspase-3/7 (+) and 7-AAD (−)) and late apoptotic/dead (Caspase-3/7 (+) and 7-AAD (+)) cells were assessed using the Muse® Cell Analyzer (Merck Millipore, Burlington, MA, USA). DNA electrophoresis was performed according to protocol described by Herrmann et al. [19 (link)].
Muse caspase 3 7 assay kit
Manufactured by Merck Group
Sourced in United States, Germany
The Muse Caspase-3/7 Assay Kit is a quantitative, fluorescence-based detection tool used to measure the activities of caspase-3 and caspase-7 enzymes in cell samples. It provides a reliable method for assessing apoptosis, a form of programmed cell death, in various experimental models and research applications.
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Lab products found in correlation
Muse cell analyzer, by Merck Group (15 mentions)
Muse annexin 5 dead cell kit, by Merck Group (4 mentions)
Muse annexin 5 and dead cell assay kit, by Merck Group (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Caspase glo 9 assay kit, by Promega (2 mentions)
Mitochondria cytosol fractionation kit, by Abcam (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Metformin hydrochloride, by Merck Group (1 mentions)
Anti mouse and anti rabbit immunoglobulin ig g horseradish peroxidase hrp linked secondary antibodies, by GeneTex (1 mentions)
Ix73 inverted microscope, by Olympus (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Muse benchtop flow cytometer, by Merck Group (1 mentions)
Epoch plate reader, by Agilent Technologies (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Spectramax m5e, by Molecular Devices (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Fitc conjugated annexin 5 and propidium iodide, by BD (1 mentions)
Muse mitopotential kit, by Merck Group (1 mentions)
Muse count viability assay kit, by Merck Group (1 mentions)
37 c incubator, by Panasonic (1 mentions)
27 protocols using muse caspase 3 7 assay kit
1
Quantifying Apoptosis via Caspase-3/7 Assay
2
Caspase-3/7 Activation and Annexin V Apoptosis Assay
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To evaluate apoptosis, the following flow cytometry experiment was performed. After 24 h of pre-incubation at 37°C, the cells were treated with various concentrations of TP4O (0, 100 or 1,000 µM). Treatment-induced caspase-3/7 activation was examined in HCT116 and RKO cells using the Muse™ Cell Analyzer (Merck KGaA) and Muse™ Caspase-3/7 Assay kit (Merck KGaA), according to the manufacturer's protocol. Following 6 h of treatment, harvested cells were mixed with the Muse™ Caspase-3/7 reagent, which contains a DNA-binding dye that is linked to a DEVD peptide substrate and a dead cell marker [7-aminoactinomycin D (7-AAD)]. Caspase-3/7 activity was detected with the fluorescence of a DNA-binding dye from the Muse™ Caspase-3/7 Assay kit and cell viability was detected with 7-AAD fluorescence using the Muse™ Cell Analyzer. The results were obtained from three independent experiments.
For the Annexin V assay, cells were treated with TP4O (0, 100 or 1,000 µM) for 12 h. Treatment-induced apoptosis was examined using the Muse™ Annexin V & Dead Cell kit (Merck KGaA) according to the manufacturer's protocol. Phosphatidylserine (PS) was detected using Annexin V, and cell viability was detected using a dead cell marker (7-AAD). The results were obtained from four independent experiments.
For the Annexin V assay, cells were treated with TP4O (0, 100 or 1,000 µM) for 12 h. Treatment-induced apoptosis was examined using the Muse™ Annexin V & Dead Cell kit (Merck KGaA) according to the manufacturer's protocol. Phosphatidylserine (PS) was detected using Annexin V, and cell viability was detected using a dead cell marker (7-AAD). The results were obtained from four independent experiments.
3
Metformin-Induced Apoptosis Mechanism
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Metformin hydrochloride, thiazolyl blue tetrazolium bromide (MTT), In Situ Cell Death Detection kit (fluorescein), compound C, carbobenzoxyvalyl-alanyl-aspartyl fluoromethyl ketone (z-VAD-fmk), and all other chemicals and reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), unless otherwise stated. All primary antibodies, anti-mouse and anti-rabbit immunoglobulin (Ig)G horseradish peroxidase (HRP)-linked secondary antibodies were obtained from GeneTex International Corporation (Hsinchu, Taiwan). Muse Caspase-3/7 Assay Kit was obtained from Merck KGaA. 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) and 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3 (link))] were obtained from Molecular Probes (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Ham’s Nutrient Mixture F12 medium, minimum essential medium, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin and trypsin-EDTA were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Mitochondria/Cytosol Fractionation Kit was bought from BioVision, Inc. (Milpitas, CA, USA).
4
Colon Cancer Cell-Fibroblast Co-Culture Assay
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The SW480, HT-29, DLD-1 and Caco-2 colon cancer cells and WI-38, CCD-18Co and BJ fibroblasts were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. For the co-culture, 30,000 SW480, HT-29, DLD-1 and Caco-2 cells were seeded onto 24-well plates with 500 µl RPMI-1640 medium. Additionally, 30,000 WI-38, CCD-18Co and BJ cells were seeded onto Transwell® inserts with 100 µl RPMI-1640 medium. After 6 h, the fibroblasts seeded onto Transwell® inserts were transferred to a 24-well plate for co-culturing with colon cancer cells. As a blank control, 100 µl RPMI-1640 medium without fibroblasts in Transwell® inserts was prepared. Media were replaced with fresh RPMI-1640 medium every 2 days. Colon cancer cell proliferation on day 5 in the presence or absence of fibroblasts was determined with a CCK-8 assay. Briefly, 50 µl CCK-8 solution was added to each well and incubated for 2 h, following which the absorbance at 450 nm was measured using a microplate reader (SpectraMax i3; Molecular Devices, San Jose, CA, USA). The apoptotic rate of co-cultured colon cancer cells was measured with a Muse® cell analyzer (Merck KGaA) using the Muse® Caspase-3/7 Assay kit. Cell images were obtained using an Olympus IX 73 inverted microscope (Tokyo, Japan).
5
Cell Viability and Apoptosis Assays
6
Apoptosis Induction by Compound 4
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The apoptosis assays were conducted using the Muse Caspase-3/7 assay kit and the Annexin V/7-AAD kit (EMD Millipore, Darmstadt, Germany) following the manufacturer’s instructions. Initially, DU-145 cells were seeded in 6-well plates at a density of 1 × 105 cells per well. After 24 h of incubation, the cells received treatment with 2.5 µM and 5 µM of compound 4 . Subsequently, the cells were incubated for 48 h. Upon completion of the treatment period, the cells were harvested using enzyme-free Cell Dissociation Buffer. The samples were then subjected to the appropriate dyes according to the manufacturer’s instructions and analyzed using the Muse Cell Analyzer (Merck Millipore, Darmstadt, Germany).
7
Caspase-3/7 Assay for Apoptosis in GBM Cells
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Muse® Caspase-3/7 Assay Kit (Merck, Germany) was used in order to verify if the analyzed compounds are able to induce apoptosis in GBM cells. This fluorescent-based assay relies on the quantitative measurements of apoptotic status based on caspase-3/7 activation analyzed simultaneously with cellular plasma membrane permeabilization and cell death.
Briefly, cells from all three cell lines (100,000 cells of A-172 and T98G cell lines, and 150,000 cells of U-138 MG cell line) were seeded on 6-well plates and incubated for 24 h. Afterward, the analyzed compounds were added in concentrations based on MTT results, and the cells were further incubated for 48 h. 0.5% or 1% DMSO and 100 nM topotecan were used as negative and positive controls, respectively. The subsequent analysis was performed on Muse™ Cell Analyzer according to the manufacturer’s recommendations (Merck, Germany).
Briefly, cells from all three cell lines (100,000 cells of A-172 and T98G cell lines, and 150,000 cells of U-138 MG cell line) were seeded on 6-well plates and incubated for 24 h. Afterward, the analyzed compounds were added in concentrations based on MTT results, and the cells were further incubated for 48 h. 0.5% or 1% DMSO and 100 nM topotecan were used as negative and positive controls, respectively. The subsequent analysis was performed on Muse™ Cell Analyzer according to the manufacturer’s recommendations (Merck, Germany).
8
Metformin-Induced Apoptosis in AGS Cells
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AGS cells (5×106 cells/75T flask) were incubated with or without 10, 20, 30 and 40 mM metformin for 48 h. The cells were collected by centrifugation at 400 × g prior to incubation with the working solution provided in the Muse Caspase-3/7 Assay Kit (Merck KGaA), according to the manufacturer’s protocol.
9
Apoptotic Cell Death Quantification
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The Muse® Cell Analyzer (EMD Millipore, Billerica, MA) and Muse® Caspase-3/7 Assay kit (cat# MCH100108) were used to determine apoptotic cell death. Live and dead cells were collected and combined and cell samples were prepared and incubated with Muse® Caspase-3/7 working solution in the dark for 30 minutes at 37 degrees. Next, the Muse® 7-AAD working solution was added for 5 minutes. Samples were read on the Muse® Cell Analyzer and results were reported as percentages of live (lower left quadrant), apoptotic (lower right quadrant), apoptotic/dead (upper right quadrant) and dead (upper left quadrant) cells.
10
Caspase-3/7 and Caspase-9 Activity Assays
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The activity of caspase-3/7 was measured according with method described previously (Adamska et al. 2019 ). Briefly, the cells were seeded in 12-well plates (1 × 105 cells/well) and treated with bersaldegenin-1,3,5-orthoacetate at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL. The concentration of DMSO added to a control sample did not exceed 0.25% (v/v). After 24 h, the cells were harvested and prepared according with the protocol of Muse Caspase-3/7 Assay Kit (Merck Millipore). Then, the cells were analysed by Muse Cell Analyzer. The experiments were performed at least in three independent repeats.
The activity of caspase-9 was measured by a luminometer. The cells were seeded in 96-well plates and exposed to bersaldegenin-1,3,5-orthoacetate at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL. The DMSO concentration in a control sample was 0.25% (v/v). The activity of caspase-9 was measured in the cells after 1, 2, 3, 4, 14, and 24 h of incubation the cells with the compound. We used Caspase-Glo 9 Assay Kit (Promega, Madison, WI, USA) and Glomax Multi + Detection System (Promega), according to the manufacturer’s instruction. The experiments were repeated three times, independently.
The activity of caspase-9 was measured by a luminometer. The cells were seeded in 96-well plates and exposed to bersaldegenin-1,3,5-orthoacetate at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL. The DMSO concentration in a control sample was 0.25% (v/v). The activity of caspase-9 was measured in the cells after 1, 2, 3, 4, 14, and 24 h of incubation the cells with the compound. We used Caspase-Glo 9 Assay Kit (Promega, Madison, WI, USA) and Glomax Multi + Detection System (Promega), according to the manufacturer’s instruction. The experiments were repeated three times, independently.
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