Cells were seeded in cell culture 10-mm × 20-mm dishes (Corning) at a density of 1 × 106 cells. After 8 h of serum starvation, the cells were treated with 5 μM and 10 μM ATRA for 24 h and 48 h. For the cell cycle analysis, 1 × 106 cells were washed in PBS and resuspended in 200 μl propidium iodide (50 μg/ml in PBS; Sigma), plus 50 μl RNaseA solution (100 μg/ml in water; Sigma) and 0/004% NP40 in PBS. The cells were incubated at 37 °C for 3 h in the dark. The cell-cycle distribution was then analyzed using flow cytometry, by fluorescence-activated cell sorting analysis (BD FACS, Canto II, BD Biosciences). The means and standard deviations were calculated from two independent experiments.
Rnase a solution
Manufactured by Merck Group
Sourced in United States, Germany
RNase A solution is a laboratory product manufactured by Merck Group. It is a purified ribonuclease enzyme used for the degradation of single-stranded RNA molecules in various experimental procedures. The solution provides a consistent and reliable source of RNase A for use in molecular biology, biochemistry, and other life science applications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (10 mentions)
Lsrfortessa, by BD (5 mentions)
Attune nxt, by Thermo Fisher Scientific (4 mentions)
Dakocytomation, by Beckman Coulter (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Cell culture 10 mm 20 mm dishes, by Corning (2 mentions)
Canto 2, by BD (2 mentions)
Flow cytometer canto 2 analyzer, by BD (2 mentions)
Prodidium iodide, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Lsr 2, by BD (1 mentions)
Annexin 5 fluos staining kit, by Merck Group (1 mentions)
Annexin 5 apoptosis detection kit 2, by BD (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Expo32, by Beckman Coulter (1 mentions)
Propidium iodide solution, by BioLegend (1 mentions)
Heraeus multifuge 1s r, by Thermo Fisher Scientific (1 mentions)
Formamide, by Merck Group (1 mentions)
36 protocols using rnase a solution
1
ATRA-induced cell cycle analysis
2
Cell Cycle Analysis by Flow Cytometry
3
Apoptosis Detection and Quantification in Cancer Cells
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Cell death induced by apoptosis was detected by AnnexinV Apoptosis Detection Kit II (BD Pharmingen™) or Annexin-V-FLUOS Staining Kit (Sigma-Aldrich) according to manufacturers’ instructions. Cells were seeded in 6-well plates and allowed to attach for 24 h. One hour after treatment with SAR (0.125 μM, 0.25 μM), cells were irradiated with 8 Gy single dose and treated with 5-aza-dC (0.1 μM, 0.5 μM) or TMZ (50 μM, 100 μM). Cells were harvested by trypsinization 4, 24, and 96 h after IR, washed twice with PBS and stained with Annexin V FITC antibody and propidium iodide. Cell staining was measured by flow cytometry (Beckman Coulter, EPICS XL). Additionally, apoptosis-induced DNA fragmentation was determined in sub-G1 fraction of cell cycle analysis (Nicoletti assay) after propidium iodide staining. Cells were treated and harvested as mentioned above, washed twice with PBS, fixed with 70% ethanol, and stored at − 20 °C overnight. After two washes with PBS cells were incubated with 0.1 mg/ml RNAseA solution (Sigma-Aldrich) at 37 °C for 20 min. Then, 50 μg/ml propidium iodide (Sigma-Aldrich) was added and the solution was incubated at 4 °C for 10 min before DNA content was measured by flow cytometry. Sub-G1 fraction was determined using EXPO32 software (Beckman Coulter).
4
Cell Cycle Analysis of iPSCs
5
FSK-Induced Cell Cycle Analysis
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HeLa cells (10 × 105) were seeded in a 60 mm Petri dish and were allowed to grow for 24 h. FSK of FSK-20 μM, FSK-40 μM, FSK-80 μM and FSK-100 μM concentrations were added to the culture medium and the cells were incubated for an additional 24 h. Thereafter, the cells were harvested with trypsin/ Ethylenediaminetetraacetic acid (EDTA), fixed with ice-cold 70% ethyl alcohol (EtOH) at −80 °C for overnight. On the next day the fixed cells were washed with PBS and incubated with 1 mg/mL RNase A solution (Sigma) at 37 °C for 30 min. Subsequently, the cells were collected by centrifugation at 2000 rpm (Heraeus Sorvall swinging bucket rotor (model #75002000), max speed: 4700 rpm, Heraeus Multifuge 1S-R, Thermo Scientific) for 5 min and further stained with 250 µL DNA staining solution (10 mg propidium iodide (PI), 0.1 mg trisodium citrate, and 0.03 mL Triton X-100 dissolved in 100 mL sterile Milli-Q water at room temperature for 30 min in the dark). The DNA content of 20,000 events was measured by flow cytometry (DAKOCYTOMATION, Beckman Coulter, CA, USA). The resultant histograms were analysed using Summit software.
6
Cell Cycle Analysis of NP Cells
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Cell cycle analysis was performed using a cell cycle measurement kit (Beyotime Institute of Biotechnology). NP cells were seeded at a density of 1x105/well in a six-well plate, pretreated with different concentrations of PCA (10 or 50 µg/ml) for 2 h and then stimulated with 200 µM H2O2 for 2 h in the presence or absence of PCA (10 or 50 µg/ml) at 37˚C. The medium was replaced with fresh culture medium, and cells were cultured for 72 h. Subsequently, the cells were washed with cold PBS and fixed with 70% ethanol at 4˚C overnight. In total, 1x105 cells were resuspended with RNase A solution (100 µl, Sigma-Aldrich; Merck KGaA) and PI (400 µl; Sigma-Aldrich; Merck KGaA) and incubated for 30 min at 37˚C. Finally, the DNA content was detected via flow cytometry (BD LSRFortessa; BD Biosciences) and the percentage of cells in each phase was analyzed by ModFit LT 5.1 (Verity Software House).
7
Cell Cycle Analysis by Nicoletti Assay
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The effects of FUS and RT on apoptosis-induced DNA fragmentation and cell cycle phase distribution were investigated by Nicoletti assay [33 (link)] using flow cytometry 4, 24, and 72 h after treatment. Briefly, cells were trypsinized from 96-well plates with 100 µL trypsin/EDTA per well and harvested into one 1.5 ml reaction tube, washed twice with PBS, and fixed with 70% ethanol at −20 °C overnight. Cells were washed again twice with PBS and incubated with 60 µL RNaseA solution (Sigma-Aldrich GmbH, Munich, Germany) at a final concentration of 0.1 mg/mL at 37 °C for 20 min. Afterwards, the DNA content was stained by propidium iodide (PI, Sigma-Aldrich GmbH, Munich, Germany) at a final concentration of 50 µg/mL at 4 °C for 5 min and cells were assessed by flow cytometry (AttuneNxT, Thermo Fisher Scientific, Darmstadt, Germany).
8
FACS Sorting and Telomere FISH Analysis
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The suspension of AT2 cells in control and G4 Tert−/− mice sorted by FACS were fixed with PFA, and dropped on slides at a concentration of 1000 cells/50 μl, then baked at 55°C overnight. Slides were washed in PBS for 15 min and then incubated in RNase A solution (Sigma) for 1 h. After washing in 2× SSC (Thermo) for 30 min, the slides were immersed in 0.005% pepsin (Sigma) for 4 min at 37°C. Then gradient alcohol dehydration was performed as followed: 1 min each in 70%, 85% and 100% alcohol. The slides were incubated in telomere PNA probe solution (Panagene), denatured at 85°C for 10 min, and then kept at room temperature for 1 h in darkness. The slides were washed in washing solution of pH 7.4 containing 20 mM Na2HPO4 (Sigma), 20 mM Tris, 60% formamide (Sigma), 0.1% μg/ml salmon sperm DNA (Sigma) and 2× SSC for 10 min. The nucleus was stained with DAPI. The sections were mounted with glycerin.
9
Cell Cycle and Apoptosis Analysis
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The cells were harvested after 48 h of reverse transfection and cell cycle assay was performed by labeling the dsDNA of the cells with PI (Propidium Iodide) (Sigma). Briefly, the harvested cells were fixed in 70% ethanol for 30 minutes at 4 °C. The cells were washed in PBS and resuspended in 1 ml of PBS. 5 μl of 10 mg/ml RNase A solution (Sigma) was added to the cells and incubated at 37 °C for 15 min. 10 μl of 1 mg/ml solution of PI was then added to the cells and incubated at 4 °C for at least 30 min before being analyzed on the flowcytometer. 20,000 events were captured on the flow-cytometer (BD LSR Fortessa, BD Biosciences) Based on the DNA content, the different phases of the cell cycle was determined by using Modfit LT 3.2 cell cycle software. Cells were harvested for Annexin V (AV) according to instructions provided by the kit manufacturer (Alexa fluor 488 Annexin V/Dead cell apoptosis kit, Invitrogen. Annexin V/Propidium Iodide (PI) staining was examined using a flow cytometer (BD LSR Fortessa). Data was analyzed with FSCExpress V6 software. Experiments were repeated three times.
10
Cell Cycle Analysis of Thyroid Cancer Cells
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Thyroid cancer cells were seeded in 6-well plates (200,000 cells per well). Twenty-four hours later, the cells were γ-irradiated and/or incubated with checkpoint inhibitors (Sigma–Aldrich): UCN‐01 (7-hydroxystaurosporin) resuspended in DMSO and caffeine (1,3,7-trimethylxanthine) dissolved in DMEM by heating (80°C) for 2 hours. After 24 hours, the cells were trypsinized and fixed in 300 µL of PBS-10% FBS and 700 µL of chilled ethanol. Following fixation overnight at -20°C, cells were incubated with RNase A solution (50 µg/mL in PBS with 0.1% Tween 20 (Sigma–Aldrich) for 30 minutes at 37°C. After suspension in propidium iodide (PI, 20 µg/L, Sigma–Aldrich), fluorescence was analyzed with a FACSCalibur flow cytometer (BD Biosciences) using BD CellQuest Pro software.
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