The MCF-7 cells were seeded at a density of 5 × 105 cells/ml in a Corning® 24-well plate. After 24 h incubation, cells were treated with either the half-maximal inhibitory concentration on cell viability (IC50) or 400 μg/ml of methanolic (MSD, MST, ML) or aqueous extracts (ASD, AST, AL) of the plant parts for an additional 24 h incubation. The cells exposed to 1 µM of staurosporine (STS) (Santa Cruz biotechnology, Dallas, TX, USA) were the positive control for the induction of apoptosis. The untreated cells, the control and unstained cells, were also used in the apoptotic status analysis. The cells were stained with fluorescein isothiocyanate (FITC)‐labeled Annexin V and phycoerythrin (PE)-labeled propidium iodide (PI), according to the manufacturer’s instructions for the FITC‐Annexin V Apoptosis Detection Kit I (Becton Disckinson Biosciences, San Diego, CA, USA). The apoptotic status of 10,000 cells were analyzed with the Becton Dickinson (BD) FACSCanto™ flow cytometer using BD FACSDiva™ 6.0 software (BD Biosciences), and characterized by Annexin V+ve/PI−ve for early apoptosis and by Annexin V+ve/PI+ve for late apoptosis. Viable and necrotic cells were also detected as characterized by Annexin V−ve/PI−ve and by Annexin V−ve/ PI+ve, respectively.
Staurosporine sts
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Staurosporine (STS) is a laboratory compound that functions as a potent and broad-spectrum protein kinase inhibitor. It is commonly used in scientific research to study cellular signaling pathways and apoptosis. STS exhibits inhibitory activity against a wide range of protein kinases, making it a valuable tool for investigating the role of kinases in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva 6, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Jetprime reagent, by Polyplus Transfection (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
3 protocols using staurosporine sts
1
Apoptosis Analysis of MCF-7 Cells
2
Cell Lysis and Apoptosis Induction
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Dulbecco’s Modified Eagle Medium (DMEM) plus GlutaMax-1 (4.5 g/L D-Glucose, 25 mM HEPES, Pyruvate), fetal bovine serum (FBS), TrypLE™ Express, and Dulbecco’s phosphate-buffered saline (PBS) were provided by Gibco® (Waltham, MA, USA). Invitrogen NP-40 cell lysis buffer was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Staurosporine (STS) was procured from Santa Cruz Biotechnology (Dallas, TX, USA). Methanol and ethanol were obtained from Honeywell Riedel-de Haen (Seelze, Germany) and Merck (Kenilworth, NJ, USA). Formic acid, dichloromethane, and chloroform were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was procured from Calbiochem (San Diego, CA, USA). The solvents are chromatography-grade or equivalent. Saudi Industrial Gas (Dammam, Saudi Arabia) provided purified carbon dioxide (CO2) gas. Ultra-pure water was produced using a Millipore (Billerica, MA, USA) system with a resistivity reading of 18.2 MΩ·cm at 25 °C.
3
Apoptosis Induction and Plasmid Transfection
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The U251 cell line was grown in Dulbecco Modified Eagle Medium (4.5 g/L glucose) supplemented with 10% fetal calf serum, antibiotics (penicillin and streptomycin) and glutamine (Life Technologies, Carlsbad, CA) in 5% CO2 at 37 °C. The HeLa cell line was grown in Dulbecco Modified Eagle Medium (1 g/L glucose) + GlutaMAX supplemented with 10% fetal calf serum, antibiotics (penicillin, streptomycin) in 5% CO2 at 37 °C. Apoptosis was induced in cultures at 70% confluency with 0.5 μg/ml staurosporine (STS; Santa Cruz, Biotechnology, Heidelberg, Germany) or etoposide 50 µg/ml (Teva Classics, Paris, France). Plasmid transfection was performed with JetPrime reagent (Polyplus) according to the manufacturer’s instructions.
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