Cell apoptosis and cell cycle analysis were examined on the Muse Cell Analyzer (EMD Millipore, Billerica, MA, USA). MDA-MB-231 cells were plated into 24-well plates at the density of 5 × 104 cells/well. Cells were incubated with MOL extract (150 µg/mL), fractions no. 1–11 (150 µg/mL), 7-octenoic acid (2.5 and 4 mg/mL), oleamide (70 and 100 μg/mL), 1-phenyl-2-pentanol (600 and 700 μg/mL), or doxorubicin (1.5 μM) for 24 h. Cell apoptosis was examined by staining with Muse™ Annexin V and Dead Cell reagent (EMD Millipore, Billerica, MA, USA; cat. no. MCH100105). For cell cycle analysis, cells were fixed in chilled 75% ethanol for 3 h at −20 °C. Then cells were washed with PBS and incubated with Muse™ Cell Cycle Reagent (Millipore, Billerica, MA, USA; cat. no. MCH100106) for 30 min in the dark at RT. Cells were read on the Muse cell analyser.
Muse annexin 5 and dead cell reagent
Manufactured by Merck Group
Sourced in United States, Germany
The Muse Annexin V and Dead Cell Reagent is a laboratory tool used to identify and quantify apoptotic and dead cells in a sample. It provides a simple and efficient method for assessing cell viability and cell death. The reagent contains Annexin V, a protein that binds to phosphatidylserine, and a DNA-binding dye that stains dead cells. This combination allows the differentiation between viable, apoptotic, and dead cells in a given cell population.
Automatically generated - may contain errors
Lab products found in correlation
Muse cell analyzer, by Merck Group (8 mentions)
Muse cell cycle reagent, by Merck Group (2 mentions)
Muse cell analyser, by Merck Group (1 mentions)
Muse cell cycle kit, by Merck Group (1 mentions)
Spectramax id3 microplate reader, by Molecular Devices (1 mentions)
Trypsin edta, by Welgene (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Muse annexin 5 and dead cell kit, by Merck Group (1 mentions)
Eclipse te2000 s, by Nikon (1 mentions)
10 protocols using muse annexin 5 and dead cell reagent
1
Apoptosis and Cell Cycle Analysis of MDA-MB-231 Cells
2
Apoptosis Assay of MDA-MB-231 Cells
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Crude hexane extract, citronellol, citronellal, or doxorubicin were treated on MDA-MB-231 cells for 24 h. Cells treat with culture medium only was used as vehicle control. Cells were harvested and transferred into a 1.5 mL microtube and washed twice in cold PBS. Then, the cells were resuspended in 1% BSA and 100 µL of Muse™ Annexin V and dead cell reagent (Merck, Darmstadt, Germany) were added to 100 µL of cell suspension (5 × 105 cells/mL) and incubated in the dark for 20 min at room temperature. The apoptotic cells were analyzed using Muse™ cell analyzer, gated on Annexin V-FITC positive cells and 7-AAD positive cells.
3
Annexin V-based Cell Apoptosis Assay
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Detection of cell apoptosis was conducted as previously described (Lu et al., 2018a (link)). Briefly, cells were digested by 0.25% trypsin and stained for 20 min in the dark at room temperature with the Muse Annexin V and Dead Cell Reagent (Merck Millipore, USA). The stained cells was analyzed by Muse Cell Analyzer (Merck Millipore, USA), at least 10,000 events were collected for the cell gate.
4
Apoptosis Analysis of D. calcarata Extracts
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The apoptosis analysis was carried out using the protocol previously described by Acikgoz et al. (2021) (link). Apoptosis was induced by seeding the cells at 1 × 105 cells/well, overnight, after which incubate them in the presence of D. calcarata extracts using the IC50 values for 24 h. The Muse® Annexin V and Dead Cell reagent (Merck-Millipore, Germany) was used to stain the cells. The cells were then placed in the dark for 20 min at room temperature. The Muse® Cell Analyser (Merck-Millipore, Germany) was used to analyse the samples.
5
Apoptosis Assay in Thyroid Cancer Cells
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A total of 7 Â 10 5 ATC cells (CAL-62 and 8505c) were seeded in 2 mL of culture medium (RPMI containing 10% FCS and 2 mmol/L L-glutamine) on 6-well plates and were allowed to adhere overnight in a humidified incubator at 37 C and 5% CO 2 . Cells were transfected the following day with siTrkA, siP75 NTR , siSort, a combination of the three siRNAs (siCombo), or control siRNA (siCont). Images were obtained after 72 hours, and viable cell number was counted using Trypan Blue with a hemocytometer. For assessing apoptosis, 1 Â 10 6 cells were incubated with 100 mL of Muse Annexin V and Dead Cell Reagent (catalog number MCH100105; Merck Millipore, Darmstadt, Germany) for 20 minutes at room temperature as per the manufacturer's instructions. The Muse Cell Analyzer (Merck Millipore) was used to determine apoptosis, and the statistics obtained revealed the percentages of the cells represented by alive, apoptotic, and dead populations.
6
Evaluating Cytotoxicity and Cell Cycle
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IndoSN-38, NaproSN- 38, Irinotecan, and SN38 were provided by Professor Jong Seung Kim, Department of Chemistry, Korea University, Seoul, Korea. Trypsin-EDTA (Welgene), FBS (Welgene), antibiotic (Welgene), MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (Amresco), Muse™ Annexin V and Dead cell reagent, Muse™ Cell cycle Kit (Merck Milipore, Billerca, MA, USA), and Caspase-3. Caspase-8 (BD biosciences, USA) were purchased. Muse™ Cell Analyzer (Merck Milipore, USA) and Spectra Max iD3 microplate reader (Molecular Devices, USA) were used.
7
Apoptosis Quantification in ATDC5 Cells
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Muse® Cell Analyzer (Millipore, USA) was adopted to assess the apoptosis in ATDC5 as per the manufacturer’s instructions. After trypsinization, the cells were harvested and subsequently washed twice with PBS. 100 μl of the Muse Annexin V and Dead Cell Reagent (Millipore, USA) was added to each sample, followed by incubation for 20 min at room temperature in the dark. The percentages of the four cell subpopulations were distinguished as follows: lower-left: viable cells, lower-right: cells in the early stages of apoptosis, upper-right: cells in the late stages of apoptosis, and upper-left: cells that have died via necrosis instead of apoptotic pathway.
8
Analyzing Cell Viability and Apoptosis
9
Erianin Induces Cell Cycle Arrest and Apoptosis in Liver Cancer
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The liver cancer cells were plated into 6-well plates at 2×105 cells/well and exposed to erianin at 40 and 80 nM for 12 h. After fixation in 70% ethanol at 4°C overnight, cells were stained with Muse™ Cell Cycle reagent (Millipore, Billerica, MA, USA) under darkness at room temperature for 30 min. Cell cycle progression was analyzed using the Muse® Cell Analyzer (Millipore, Billerica, MA, USA).
The liver cancer cells were plated into 6-well plates at 2×105 cells/well and exposed to erianin at 40 and 80 nM for 24 h. Cells were stained with Muse™ Annexin V and Dead Cell reagent (Millipore, Billerica, MA, USA) under darkness at room temperature for 20 min. Cell apoptosis was analyzed using the Muse® Cell Analyzer (Millipore, Billerica, MA, USA).
The liver cancer cells were plated into 6-well plates at 2×105 cells/well and exposed to erianin at 40 and 80 nM for 24 h. Cells were stained with Muse™ Annexin V and Dead Cell reagent (Millipore, Billerica, MA, USA) under darkness at room temperature for 20 min. Cell apoptosis was analyzed using the Muse® Cell Analyzer (Millipore, Billerica, MA, USA).
10
Apoptosis and Mitochondrial Integrity Analysis
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HepG2 and SMMC-7721 cells were seeded into six-well plates at a concentration of 3×105 cells/well, and incubated for 24 h. The cells were then exposed to ERN, Tf-LP, LP-ERN or Tf-LP-ERN (to a final ERN concentration of 10 nM) for a further 24 h, and then incubated with 100 μL of Muse™ Annexin V and Dead Cell reagent (Millipore, Billerica, MA, USA) under darkness for 20 min at 25°C. Cell apoptosis was then analyzed using the Muse® Cell Analyzer (EMD Millipore, Billerica, MA, USA).
Another set of six-well plates seeded with HepG2 and SMMC-7721 cells were treated with ERN, Tf-LP, LP-ERN or Tf-LP-ERN (to a final ERN concentration of 10 nM) for 12 h, and then incubated with 10 μM of 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Calbiochem, San Diego, CA, USA) for 20 min at 37°C. The changes in the intensity of red and green fluorescence of cells were observed using a fluorescence microscope (Eclipse TE 2000-S, Nikon Corp., Tokyo, Japan).
Another set of six-well plates seeded with HepG2 and SMMC-7721 cells were treated with ERN, Tf-LP, LP-ERN or Tf-LP-ERN (to a final ERN concentration of 10 nM) for 12 h, and then incubated with 10 μM of 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Calbiochem, San Diego, CA, USA) for 20 min at 37°C. The changes in the intensity of red and green fluorescence of cells were observed using a fluorescence microscope (Eclipse TE 2000-S, Nikon Corp., Tokyo, Japan).
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