Cultured cells were harvested and washed with 1× PBS. The cells were then fixed with 70% ethanol for 10 min. Further, cells were washed, re-suspended in PBS containing RNAase (Sigma Aldrich, St. Louis MO, USA) (100 μg/ml), and incubated for 30 min at 37 °C. Thereafter, propidium iodide (50 μg/ml) was added and the cells were immediately acquired on FACS Calibur (BD, San Jose, CA, USA).
Rnaase
Manufactured by Merck Group
Sourced in United States, Germany
RNAase is a laboratory enzyme that catalyzes the hydrolysis of ribonucleic acid (RNA) molecules. It is commonly used in molecular biology and biochemistry research to remove RNA from samples, preparing them for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (23 mentions)
Facscalibur, by BD (4 mentions)
Hepes, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Facscan, by BD (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Nystatin, by Merck Group (2 mentions)
Penicillin g, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Propidium iodide, by BD (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Topotecan hydrochloride, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Facsaria 2 cytometer, by BD (1 mentions)
Ethanol, by Avantor (1 mentions)
Fitc labeled annexin 5, by BD (1 mentions)
Fcs express v3, by BD (1 mentions)
39 protocols using rnaase
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by FACS
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Fluorescence activated cell sorting (FACS) cell cycle analysis was performed using the BD Brdu FitC kit according to manufacturer instructions (BD). Cells were collected on a LSR II flow cytometer and the analysis was performed using FlowJo software. DNA content analysis was performed by staining cells with propidium iodide (PI) (Sigma) and analysis by FACS. Briefly, cells were trypsinized, washed in PBS, and fixed overnight at 4 degrees Celsius in 70% ethanol. Cells were washed in PBS and incubated at room temperature with 1 mg/ml RNAase (Sigma) in PBS for 1 hour before treatment with treatment 20 ug/ml PI (Sigma). At least 10,000 events were collected for analysis. Cells were analyzed using FlowJo data analysis software.
3
Apoptosis Quantification in Liver Cells
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The cultured L02 cells (5×105 cells each group) or cell suspensions from homogenized middle left liver tissues were fixed in ice-cold 70% ethanol and centrifuged to collect a cell pellet that was resuspended in phosphate-buffered saline (PBS). After washing, cells were incubated with RNAase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in PBS for 30 min at 37°C. After further washing, pellets were resuspended in 0.5 ml propidium iodide staining solution (50 µm/ml) and incubated for 30 min at 4°C. After filtering through a nylon mesh (pore size, 48 µm), apoptosis was detected using a flow cytometer (FACScalibur; BD Biosciences, San Jose, CA, USA) at an excitation setting of 488 nm.
4
Topotecan and Safranal Cytotoxicity Assay
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Topotecan hydrochloride (TPT) and safranal (SAF) (Sigma-Aldrich, Missouri, United States) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Missouri, United States), the stock solution of TPT was kept at −20°C. In preparation for an experiment, a serial dilution of TPT was prepared in the medium to achieve a concentration range of 0.001–1 μM and the concentration of DMSO in all samples was 0.02%. Primary monoclonal antibodies against γ-H2AX and TDP1 and secondary antibodies (anti-rabbit and anti-mouse) were obtained from Cell Signaling Technology (Danvers, MA, United States). In addition, propidium iodide (PI), RNAase, and crystal violet were purchased from (Sigma-Aldrich, Missouri, United States).
5
Cell Cycle Analysis by Flow Cytometry
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Cell cycle distribution was analyzed with flow cytometry. Harvested cells were fixed in 70% precooled ethanol. Then, cells were treated with RNAase (0.1 mg/mL; Sigma-Aldrich Co, St Louis, Missouri) for 30 minutes and incubated with 1 mL of protease inhibitor (50 µg/mL; Sigma-Aldrich Co). Finally, flow cytometry analysis (BD Biosciences, San Jose, California) was performed to assess cell cycle distribution. Three independent experiments were conducted.
6
In Situ Hybridization Protocol for Tissue Sections
7
Cell Cycle Dynamics of Cervical Cancer
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We observed a decrease of cell proliferation in HeLa and INBL cell lines at 48 hours; therefore, we decided to determine the phases of the cell cycle at these time points. To determine the cell-cycle phases, 1 × 106 HeLa and INBL cervical cancer cells were seeded and incubated with 100 IU/ml IL-2 for 24 and 48 hours. Cell-cycle determination was performed using the propidium iodide incorporation technique. Cells were fixed using 70% ethanol (JT Baker, USA) for 12 hours, and RNA was removed by incubating with an RNAase (Sigma, USA) for one hour at 37°C. Cells were incubated with 3 μL of propidium iodide (Sigma; 50 mg/mL) for 10 minutes. Cell-cycle phases were determined using flow cytometry on a FACSAria II cytometer (BD, USA). Data were analysed using FCS express 7 De Novo software. To determine if the observed cell arrest is permanent or transient, after 48 hours of cell culture in the presence of IL-2 the medium was removed, and fresh medium without IL-2 was added, leaving the cells in culture for 24 and 48 hours to analyse the phases of the cell cycle.
8
Evaluating miR-18a's Impact on Apoptosis and Cell Cycle
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The impact of miR-18a on cell apoptosis and the cell cycles of HepG2 and SMMC7721 cells were analyzed with flow cytometry. For apoptosis experiments, cells were harvested and resuspended in 200 µL PBS. Propidium iodide (PI) and FITC-labeled Annexin V (BD Biosciences) were then added into the mixture and incubated for 30 minutes at room temperature. For cell cycle assay, the harvested cells were fixed in 70% pre-cooled ethanol. The samples were taken before DNA staining, and the supernatant was removed by centrifugation. After the samples were treated with RNAase (0.1 mg/mL; Sigma-Aldrich Co., St Louis, MO, USA) for 30 minutes, 1 mL of PI (50 µg/mL; Sigma-Aldrich Co.) was added, and flow cytometry analyses was performed to assess the distribution of cells in each phase of the cell cycle.
9
Cell Cycle and Apoptosis Analysis
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Single-cell suspensions were fixed in 70% precooling ethanol overnight at 4 °C, washed twice with PBS, and incubated with 1 mL propidium (PI, 50 mg/L)/RNAase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 min under dark conditions. Cells were then evaluated using a flow cytometer (Gallios, Beckman Coulter, Shanghai, China) at 488 nm. To analyze apoptosis rates, the cell suspension was incubated with 10 μL Annexin V-fluorescein isothiocyanate (FITC) and 5 μL PI without light exposure for 15 min and were analyzed immediately with the use of a flow cytometry.
10
In Situ Hybridization Tissue Imaging
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Procedures for In situ hybridization were carried out similarly as described [6 (link), 7 (link)]. Tissue sections were cut at −20 °C, and then fixed with 4 % paraformaldehyde, followed by three washes of 0.1 M phosphate buffer, air-dried, and stored at −20 °C until use. For In situ hybridization, sections were dried at room temperature, followed by pretreatment of proteinase K (1 μg/ml). Sections were then air-dried and hybridized with S [35 (link)]-labelled riboprobes by incubation at 60 °C for 18 h. After hybridization, tissue sections were treated with RNAase (20 μg/ml) (Sigma-Aldrich, St. Louis, MO), decreasing salinity washes and high stringency (68 °C) wash. After dehydration and air-drying, tissue sections were exposed to Kodak Biomax film. Images were captured with image analysis system (MCID, Imaging Research, Ontario, Canada).
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