48 h after transfection, the cells were harvested, washed with PBS and fixed with 70% ethanol for 4 h. The fixed cells were resuspended in PBS containing RNase A (10 mg/ml; BD Biosciences) and stained with PI in dark at room temperature for 30 min. Cell cycle distribution was detected and analyzed by FACScan flow cytometer.
Rnase a
Manufactured by BD
Sourced in United States, United Kingdom
RNase A is a ribonuclease enzyme that catalyzes the hydrolysis of single-stranded RNA molecules. It is commonly used in molecular biology and biochemistry applications to remove unwanted RNA from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (14 mentions)
Facscalibur, by BD (8 mentions)
Facscalibur flow cytometer, by BD (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
Facscan, by BD (3 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (3 mentions)
Triton x 100, by BD (3 mentions)
Cellquest, by BD (3 mentions)
Np 40, by BD (3 mentions)
Cycletest plus dna reagent kit, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Annexin 5 apc, by Elabscience (2 mentions)
Fascanto 2, by BD (2 mentions)
Propidium iodide staining buffer, by BD (2 mentions)
Modfit software v3, by Verity Software House (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Modfit lt software, by Verity Software House (2 mentions)
Annexin 5 fitc, by BD (2 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
72 protocols using rnase a
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Fixation and Propidium Iodide Staining
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Cells were collected after digestion and then prepared as a single-cell suspension at a concentration of 1×105 cells/mL, and then the cells were fixed in pre-cooled 75% ethanol and placed in a refrigerator at 4°C overnight. Before flow cytometry determination, cells were washed twice with PBS to remove the fixative. After adding 100 μL of RNaseA (BD Biosciences, San Jose, CA, USA), cells were protected from the light in a 37°C water bath, and 30 min later, 400 μL of PI (propidium iodide) was added to stain the cells for 30 min at 4°C in the dark.
3
Cell Cycle Analysis by Flow Cytometry
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The cells were harvested and fixed with 70% prechilled ethanol for > 24 hours at −20°C. The fixed cells were washed twice with phosphate-buffered saline, then treated with 100 μg/mL RNase A and 50 μg/mL PI mixed buffer (BD Biosciences, San Jose, California) for 30 minutes at 37°C. The cell cycle distribution was analyzed with Modfit software (Verity Software, Topsham, Maine) from the histogram of the DNA content measured with a flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, New Jersey). The increased G2/M cells (%) were used to quantitate the induction of the G2/M block, which was obtained by subtracting the number of control G2/M cells from those of the irradiated samples.5
4
Cell Cycle Analysis of Pancreatic Cancer Cells Treated with KL-6
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PANC-1 cells were seeded in a 10 cm cell culture dish at a density of 2 × 106 cells per dish and incubated for 24 h. The culture medium was then replaced with fresh medium containing different concentrations of compound KL-6 (0, 0.5, 1, 2 µM). After incubating for 24 h, the cells were trypsinized, collected, and fixed with ice-cold 95% ethanol overnight at 4 °C. Following fixation, the cells were washed with PBS and incubated with propidium iodide (PI) staining solution containing RNase A (BD Biosciences, San Jose, CA, USA) for 30 min at 37 °C in the dark. The cell cycle distribution was determined using a flow cytometer, and the data were analyzed using FlowJo software. The percentage of cells in the G0/G1, S, and G2/M phases was quantified to assess the cell cycle arrest induced by compound KL-6 (Zhang et al., 2019 (link)).
5
Cell Cycle and Apoptosis Analysis
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Cell cycle inhibition was determined using the Cycle Test Plus DNA Reagent Kit (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA), according to the manufacturer’ s instructions. The treated cells were collected 48 h after transfection and washed twice with phosphate buffer solution (PBS). The cells were fixed with ice-cold 70% ethanol and incubated at −20 °C overnight. Subsequently, the fixed cells were washed twice with PBS and incubated with 200 μL propidium iodide (PI) solution with RNaseA (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA) at room temperature for 30 min in the dark. The treated cells were subsequently analyzed using the NovoCyte Flow Cytometer (ACEA Biosciences, Inc, San Diego, CA, USA) with NovoExpress® software (1.3.0; ACEA Biosciences, Inc, San Diego, CA, USA, 2018). Early and late apoptotic cells were analyzed using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA). The cells were trypsinized without ethylenediamine tetraacetic acid and scraped twice with cold PBS. Subsequently, the cells were stained using the Annexin V-FITC/PI kit at room temperature for 30 min in the dark according to the manufacturer's protocol. The treated cells were analyzed by the procedure used for the cell cycle analysis.
6
Flow Cytometric Analysis of BMSCs
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Harvested washed one million BMSC suspended in 1mL PBS were fixed by adding them slowly drop by drop in 9mL 75% ethanol while vortexing. Cells in 75% ethanol were incubated overnight at 4°C and then the pallet was reconstituted after centrifugation in 200μL of a mixture containing 20 μg/mL propidium iodide (PI; BD Biosciences, USA), and 10 μg/mL RNase-A (Coolaber, China) in 0.2% Triton-x water. The stained cells were incubated 15 minutes at RT° and then analyzed using flow cytometry Accuri C6 (BD Biosciences, San Jose, CA, USA).
7
Apoptosis and Cell Cycle in TBMS1 Treatment
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Cells (2×105 per well) were plated into 6-well plate and cultured with different concentrations of TBMS1 for 24 h. Cells were harvested and washed in PBS (phosphate buffered solution). For apoptosis analysis, cells were tested using an apoptosis kit (BestBio, Shanghai, China) and analyzed by flow cytometry (BD Biosciences, CA, USA). For cell cycle analysis, cells were fixed in 70% ethanol at 4ºC overnight. After washing with phosphate-buffered saline (PBS), the fixed cells were incubated in PBS containing 20 μg/mL of propidium iodide (PI), 200 μg/mL of RNase A, and 0.1% Triton X-100 (BD Biosciences, CA, USA) at 37ºC for 30 minutes. The stained cells were then analyzed for cell cycle distribution using a FACSCalibur flow cytometer (BD Biosciences, CA, USA).
8
Cell Cycle Analysis of Thiamine Derivatives
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A549 cells were treated with different dosages of Thiamine (10 μM) or Oxythiamine (0–100 μM) for 24 and 48 h. All the cells were trypsinized, washed and then fixed in 70% ethanol at a density of 1 million cells/ml before preserving in − 20 °C for no less than 24 h. After overnight incubation at 4 °C in ethanol, cells were washed and suspended in 0.5 mL DNA staining solution [50 μg/ml PI and 100 μg RNaseA in PBS (BD, USA)] 15 min at room temperature before flow cytometry. Finally, samples were analyzed by Cell-Quest software (Becton–Dickinson). The experiments were carried out three times on separated days.
9
Cell Cycle Analysis of A375 and A431 Cells
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A375 and A431 cells were seeded and treated with LCH (0, 10, 20, and 30 μM) for 48 h. After incubation, cells were harvested and fixed with 70% ethanol at –20°C for 2h. The cells were washed with 1×PBS, and stained with RNase A and propidium iodide (PI; BD Biosciences, Piscataway, NJ, USA). Finally, samples were incubated at 37˚C for 30 min in the dark. After reaction with reagent, they were analyzed using a fluorescence-activated cell sorting (FACS; BD Biosciences) according to the manufacturer’s instructions.
10
Cell Cycle Analysis via Flow Cytometry
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After treatment with BI2536, DDP or their combination for 24 h, 1 × 106 cells were collected, trypsinized, and fixed in 70% ethanol overnight. Then, the cells were washed three times with pre-cooled PBS and incubated with a PI-staining solution with RNase A (BD Biosciences, America) for at least 15 min at room temperature before analysis. The cells were run on a FACScan cytometer (BD Biosciences, America) in accordance with the manufacturer's guidelines.
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