At 48 h after transfection, flow cytometry was applied to analyze the MCF-7 cell cycle distribution. Briefly, the transfected MCF-7 cells were collected by trypsinization and then fixed with ethanol overnight at 4°C. Subsequently, the MCF-7 cells were washed by PBS and stained with ribonuclease A (100 µg/ml) and propidium iodide (PI; 50 µg/ml; both Beyotime Institute of Biotechnology, Shanghai, China) in the dark at 4°C for 30 min. Finally, a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the cell cycle distribution. Tests were repeated at least three times.
Ribonuclease a
Manufactured by Beyotime
Sourced in China
Ribonuclease A is a protein-based enzyme that catalyzes the hydrolysis of ribonucleic acid (RNA). It is commonly used in molecular biology and biochemistry laboratories for the degradation and purification of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Beyotime (3 mentions)
Facscanto 2, by BD (1 mentions)
Annexin 5 fluorescein isothiocyanate propidium iodide apoptosis detection kit, by BD (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Rnase a, by Beyotime (1 mentions)
Perfection v600 scanner, by Epson (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Biospot version 5, by Cellular Technology (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
P4170, by Merck Group (1 mentions)
Modfit software, by Verity Software House (1 mentions)
Epics xl flow cytometer, by Beckman Coulter (1 mentions)
6 protocols using ribonuclease a
1
Cell Cycle Analysis of Transfected MCF-7 Cells
2
Apoptosis Evaluation of OSCC Cells
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5-ALA, fetal bovine serum (FBS), 2′,7′-dichlorodihydrofluorescein diacetate, and MTT (Sigma) were used. Our study also utilized the following materials and instruments: enzyme labeling instrument produced by Bio-Rad Biomedical Products (Shanghai) Co., Ltd., Dulbecco’s modified Eagle medium (DMEM) by Shanghai Yuanpei Biotechnology Co., Ltd., semiconductor laser by Beijing Bingshou Technology Co., Ltd., annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit by BD Co., flow cytometer by BD Co.; ribonuclease A by Beyotime Biotechnology Co., Ltd., human OSCC cell line SCC25 from ATCC that was cultured at 37 °C, 5% CO2.
3
Cell Cycle Analysis of Arsenite-Exposed HaCaT Cells
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The cell cycle was detected by the cell cycle test kit. As per the manufacturer’s instructions, synchronizing the cells before exposure required 48 h of culture in DMEM with 0.5% FBS. After continuous exposure to arsenite over a period of time, HaCaT cells were harvested at different passages, collected with 0.25% trypsin without EDTA, and washed twice with 4 °C pre-cooled PBS. The cells were left at −20 °C overnight with the addition of 1 mL of 70% pre-cooled ethanol. After two rounds of washing in cold PBS, the cells were stained for 30 min with 20 μg/mL of PI (Beyotime Institute of Biotechnology, Haimen, China) and 200 μg/mL of ribonuclease A (RNaseA, Beyotime Institute of Biotechnology, Haimen, China) diluted in PBS. Following the above processing, the data were analyzed using cell cycle analysis software based on flow cytometry (Beckman Coulter FC500, Brea, CA, USA) for detection. The experiment was repeated three times.
4
Evaluating Cancer Cell Clonogenicity
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To investigate the colony-forming ability of cancer cells, 100 cells were seeded into 12-well plates and incubated for 7 days in an incubator at 37°C with 5% CO2. The cells were subsequently fixed with 75% ethanol for 20 min at room temperature and stained using crystal violet (5 g/l) for 20 min at room temperature. Cell colonies in each groups were imaged using an Epson Perfection V600 scanner (Seiko Epson Corporation, Suwa, Japan) and the results were analyzed using BioSpot® version 5.0 software (Cellular Technology Limited, Cleveland, OH, USA).
To examine cell proliferation, the proliferation index of each group was assessed using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) as previously described (29 (link)). The proliferation index was calculated as the absorbance detected in the experimental group-the absorbance detected in the blank group.
To analyze the cell cycle, 5×106 cells were fixed with 70% ethanol for 30 min at 4°C. The cell samples were stained with 200 µl propidium iodide (PI; Beyotime Institute of Biotechnology, Haimen, China) in a solution containing ribonuclease A (Beyotime Institute of Biotechnology) for 10 min at room temperature. Subsequently, the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo 7.6.1 (FlowJo LLC, Ashland, OR, USA).
To examine cell proliferation, the proliferation index of each group was assessed using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) as previously described (29 (link)). The proliferation index was calculated as the absorbance detected in the experimental group-the absorbance detected in the blank group.
To analyze the cell cycle, 5×106 cells were fixed with 70% ethanol for 30 min at 4°C. The cell samples were stained with 200 µl propidium iodide (PI; Beyotime Institute of Biotechnology, Haimen, China) in a solution containing ribonuclease A (Beyotime Institute of Biotechnology) for 10 min at room temperature. Subsequently, the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo 7.6.1 (FlowJo LLC, Ashland, OR, USA).
5
Cell Cycle Analysis of BGC-823 and SGC-7901 Cells
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BGC-823 and SGC-7901 cells were collected by centrifugation at low speed for five minutes, followed by adequate washing with phosphate buffer, and then, the cells were fixed with 70% ethanol, incubated for 12 h at 4°C in an ice bath, and stained with Ribonuclease A (ST576, Beyotime, Shanghai, China) and PI (P4170, Sigma Aldrich, USA) staining buffer. The cells were suspended in staining solution (P4170, Sigma Aldrich, MS, USA), and the cell cycle was analyzed by flow cytometry (BD, Biosciences, NJ, USA). The Modfit software (Verity Software House, GA, USA) provided the estimation of the percentage of cells in G0/G1, S, and G2/M phases of the cycle.
6
Cell Cycle Analysis of A375/B16 Cells
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A375/B16 cells were treated with different concentrations of cinobufagin for 24 h, and then collected by digestion and made into cell suspensions. The cells were fixed in pre-cooled 70% ethanol solution at 4°C for 2 h and then centrifuged at 800× g in a low temperature centrifuge for 5 min. The ethanol solution was discarded and the cells were washed twice with PBS. Then they were precipitated and suspended in 500 μL phosphate buffer containing 0.02 mg/mL propidium iodide (PI) (Cat no. ST512; Beyotime, Shanghai, China) and 0.1 mg/mL Ribonuclease A (Cat no. ST576; Beyotime, Shanghai, China). After 30 min incubation in the dark at 37°C, the cells were precipitated and suspended in phosphate buffer solution. The Epics XL Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) was used to detect fluorescence of the PI-DNA complex, and Winmdi 2.8 software (Scripps Research, La Jolla, CA, USA) was used to analyze the cell distribution at different stages of the cell cycle (25 (link)).
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