Cells were harvested at 48 h after transfection. The cells were washed with PBS and fixed in ethanol at −20°C. The cells were then washed with PBS, rehydrated, and resuspended in propidium iodide (PI)–RNase A solution (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. The stained cells (1 × 105) were then analyzed for DNA content with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Pi rnase a solution
Manufactured by Merck Group
Sourced in United States, Finland
The PI)-RNase A solution is a laboratory reagent used for the digestion and removal of RNA molecules from DNA samples. It contains the enzyme RNase A, which selectively hydrolyzes single-stranded RNA, leaving the DNA unaffected. This solution is commonly used in DNA purification and analysis workflows to eliminate potential RNA contamination.
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6 protocols using pi rnase a solution
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by Flow Cytometry
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After a specific incubation time with the Sm4 inhibitor, cells were harvested using Trypsin-EDTA solution (0.25%, Sigma Aldrich, St. Louis, MO, USA) and then centrifuged. The cells were washed twice with cold PBS, suspended in 2 mL of 70% ice-cold ethanol solution for fixation, and subsequently centrifuged. The cells were then washed twice with cold PBS. Thereafter, the pellets were suspended and incubated in 300 µL of propidium iodide (PI)-RNAse A solution (Sigma Aldrich; Merck KGaA, St. Louis, MO, USA) for 30 min at 37 °C in the dark. DNA content was analysed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The obtained FCS files of PI spectra were assessed using ModFit LT 5.0 software (Verity Software House, Topsham, ME, USA). The experiments were performed in triplicates.
3
Cell Cycle Analysis by Flow Cytometry
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At 48 h post-transfection, we collected SGC-7901 and MGC-803 cells, washed them by PBS, and then fixed them with ethanol under − 20 °C. After rinsing by PBS again, cells were subjected to rehydration and resuspension for 30 min into the 10 µl propidium iodide (PI)-RNase A solution (Sigma-Aldrich; Merck KGaA) under 37 °C. After staining, we used a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) for measuring DNA content in 1 × 105 cells. Finally, we utilized FlowJo 7.6.1 software (FlowJo LLC, Ashland, OR, USA) for result analysis.
4
Cell Cycle and Apoptosis Analysis
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H358, H292 and A549 cells were seeded to 6-well plates (5 × 105 cells/well) and cultured to 60–70% confluence. H358 and H292 cells were transfected with siRNA-2/siNC, while A549 cells were transduced with overexpression/vector virus and treated with DMSO/2 mM MK-2206. At 48 h after treatment, lung cancer cells were harvested, washed with ice-cold PBS and stained according to the manufacturers’ instructions. For cell cycle distribution analysis, the cells were fixed in cold ethanol overnight, rehydrated and stained with propidium iodide (PI)-RNase A solution (Sigma) in the dark at 37°C for 30 min. For apoptosis analysis, the cells were labeled with Annexin V-fluorescein isothiocyanate (FITC) and PI (Beyotime) in the dark at 4°C for 20 min. DNA content and cell apoptosis was then analyzed by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
5
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested at 48 h after siRNA transfection. The cells were washed with PBS and fixed in ethanol at −20°C. The cells were then washed with PBS, rehydrated and resuspended in propidium iodide (PI)-RNase A solution (Sigma) at 37°C for 30 min. The stained cells (1 × 105) were then analyzed for DNA content with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
6
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested at 48 hours after transfection. The cells were washed using PBS and fixed in ethanol at −20°C. The cells were then washed with PBS, rehydrated and resus-pended in propidium iodide (PI)-RNase A solution (Sigma, Finland) at 37°C for 30 minutes. The stained cells (1×105) were then analyzed for DNA content using a flow cytometer (BD Biosciences, San Jose, CA, USA).
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