Flow cytometry was performed as previously described [24 (link)]. Following treatment, cells were harvested on ice and fixed in 70% ethanol for at least 1 hr at −20°C. Thereafter, cells were washed in PBS and resuspended in 50 μg/mL propidium iodide/RNAse A solution (0.5 mL per 106 cells) (Sigma-Aldrich) for 30 min in the dark at room temperature. The percentage of cells in different phases of cell cycle was analysed with a Beckman Coulter Cytomics FC500 flow cytometer.
Propidium iodide rnase a solution
Manufactured by Merck Group
Sourced in United States
Propidium iodide/RNAse A solution is a laboratory product that contains a combination of propidium iodide and RNAse A. Propidium iodide is a fluorescent dye that binds to DNA, while RNAse A is an enzyme that degrades RNA. This solution is commonly used in cell biology and flow cytometry applications.
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2 protocols using propidium iodide rnase a solution
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Synchronization and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HL-1 and NIH3T3 cells were initially plated at a density of 5 x 10 5 cells/well in 3.5 cm dishes. Next day cells were transfected with PDE5A isoforms GFP-tagged vectors. Twenty four hours later, cells were synchronized with 0.5 mM L-Mimosine (Sigma-Aldrich, CA, USA). After 16 hrs, cells were washed with PBS and incubated in fresh culture media. At various times after release from the L-Mimosine block the cells were treated with trypsin, harvested, and centrifuged at 2000 g for 5 min.
The pellets were washed with PBS and then centrifuged again. Cells were fixed with 1% w/v paraformaldehyde for 1hr at 4°C, then centrifuged for 5 min at 300 g; supernatant was removed by aspiration and pellets washed with PBS. For cell membrane solubilization, 1 ml 70% v/v ethanol was added to the cell pellet, and cell suspension incubated overnight at 4°C. Fixed cells were washed with PBS, centrifuged and incubated for 3 hr at room temperature with propidium iodide/RNAse A solution (PI, 50 μg/mL, Sigma-Aldrich, CA, USA), ribonuclease (1 mg/ml Sigma-Aldrich, CA, USA). The cell cycle distribution and DNA content were measured using a CyAn cytofluorimeter (Dako). 5 x 10 3 cells were counted for each sample. The percentage of cells in different phases of the cell cycle was analyzed by FlowJo (TreeStar, Ashland, OR, USA).
The pellets were washed with PBS and then centrifuged again. Cells were fixed with 1% w/v paraformaldehyde for 1hr at 4°C, then centrifuged for 5 min at 300 g; supernatant was removed by aspiration and pellets washed with PBS. For cell membrane solubilization, 1 ml 70% v/v ethanol was added to the cell pellet, and cell suspension incubated overnight at 4°C. Fixed cells were washed with PBS, centrifuged and incubated for 3 hr at room temperature with propidium iodide/RNAse A solution (PI, 50 μg/mL, Sigma-Aldrich, CA, USA), ribonuclease (1 mg/ml Sigma-Aldrich, CA, USA). The cell cycle distribution and DNA content were measured using a CyAn cytofluorimeter (Dako). 5 x 10 3 cells were counted for each sample. The percentage of cells in different phases of the cell cycle was analyzed by FlowJo (TreeStar, Ashland, OR, USA).
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