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19 protocols using rnaase a

1

Cell Cycle Analysis of Irradiated Cells

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Cells were cultured for 36–40 h before irradiation, and were harvested by trypsinization at 48 h following irradiation. The cell number was enumerated with the help of a cell hemocytometer. The extent of proliferation was assessed by calculating the proliferation index (Px) as follows:
Proliferation index Px = Nt / N0;
where Nt is the cell number at different post-irradiation times and N0 is the number at the time of plating. Cells were fixed with ice-cold 80% ethanol and stored at 4°C for at-least 24 h before analyzing the DNA content by flow cytometry. Ethanol fixed cells were washed twice with PBS and incubated with RNAase-A (cat# 10109142001, Sigma, USA) for 45 minutes at 37°C. Cellular DNA was stained with propidium iodide (25 µg/mL) (0.1 x 106 cells/100µL of PBS) and incubated in dark at room temperature for 30 minutes. PI-stained cells were then subjected to flow cytometry with CytoflexS (Beckman Coulter, California, USA). All the data was acquired with CytExpert software and was provided with a cytometer. Acquired data were analyzed with the FlowJo_10.4 version. The percentages of cells in G0/G1-, S-, and G2+M phases were determined, after filtering for doublets and aggregates using the inbuilt software.
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2

Protosteloid Amoebae DNA and RNA Isolation

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Chromosomal DNA was isolated from liquid cultures of protosteloid amoebae. Adherent cells were washed in phosphate buffer and harvested by centrifugation at 200 x g which was sufficient to deplete nearly all residual yeast cells. Trophozoites were lysed in 50 mM Tris–HCl (pH 7.5) with 2% [w/v] of SDS and 0.5 mM Na2EDTA, followed by a 1-h incubation at 60°C with RNAase A (Sigma–Aldrich, Taufkirchen, Germany) and Proteinase K (Sigma–Aldrich) at final concentrations of 100 and 200 µg ml−1, respectively. Further purification steps like extractions with phenol-chloroform and precipitation with isopropanol were carried out as described (Sambrook and Russell 2001 ).
For the isolation of total RNA, cells were rapidly harvested from the agar surface, resuspended in 2 ml of phosphate buffer, and centrifuged for 1 min at 6,000×g. Pellets were shock-frozen in liquid N2 and stored −80°C. RNA was prepared by phenol extraction with subsequent additions of 500 µl of TRIsure (Bioline, Luckenwalde, Germany) and 200 µl of chloroform. Phase separation was achieved by centrifugation followed by an additional extraction with 500 µl of chloroform. Precipitation was as for DNA. RNA quantity and impurities were determined spectrophotometrically. Integrity of the isolated RNA was checked by agarose gel electrophoresis
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3

Cell Cycle Analysis of MDA-MB-231 Cells

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Cell cycle distribution of MDA‐MB‐231 cells was measured with flow cytometry using the nuclear stain propidium iodide (PI) (Sigma Aldrich). Briefly, cells (7.5 x 105) were seeded into T25 flasks and allowed to adhere overnight. After incubating cells with GEM and DOX solutions for 24–72 hr, adherent cells were collected with trypsin and washed 2x in PBS (5 ml). Cells were then fixed by slowing adding 70% (v/v) ethanol (5 ml) into a concentrated cell suspension (0.5 ml). After incubating the cells in ethanol for at least 1 day at 4°C, the cells were washed 2x in PBS through repeated centrifugation steps. The cells were then stained in a PBS solution (0.5 ml) containing Tween 20 (0.01%, v/v), 100 µg/ml of RNAase A (Sigma Aldrich), and PI (10 µg/ml) for 30 min at room temperature in the dark. After staining, cells were kept on ice and fluorescence was quantified using a Becton Dickinson FACSAria cell sorter with a 633‐nm laser with 610/20 PMT.
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4

Cell Cycle Analysis by Flow Cytometry

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Cell cycle was analysed by flow cytometry [42 (link)]. Cells were plated in 6 wells at a density of 1.5 × 105 cells. After the treatments, cells were trypsinized, washed with PBS, permeabilized with 70% ice cold ethanol, washed again with PBS and incubated with propidium iodide and 100 μg/ml RNAase A (Ribonuclease A from bovine pancreas R6513-10MG Sigma-Aldrich, St Louis, USA) for 20 min. When GFP population was examined, cell fixation before permeabilization was required, as previously described by Lamm et al [42 (link)]. Cells were analysed on a FACScan using CellQuest software, and cell cycle was determined using FlowJo software. In the case of PTEN overexpression, GFP population was gated in order to analyze cell cycle in a 100% transfected population.
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5

Cell Cycle and Apoptosis Analysis

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Cells with different treatments were trypsinized, washed with PBS, and fixed in ice-cold 70% ethanol at 4 °C overnight. After being washed with PBS, cells were incubated with RNAase A (Sigma) in PBS for 30 min at 37 °C and then stained with 50 mg/ml propidium iodide. Cell cycle data were collected with FACS Calibur (Becton Dickinson) and analyzed with FlowJo software. Apoptosis of cells was analyzed with FACS using Cells Annexin V Apoptosis Detection Kits as per the manufacture’s standard procedures (Affymetrix eBioscience).
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6

Flow Cytometry Analysis of Taraxerol Acetate

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Flow cytometry was utilized to assess the effect of taraxerol acetate, and data were analyzed using the FACSCalibur platform equipped with CellQuest software. The ModFit LT cell cycle analysis software (version 4.0; Verity Software House, Inc., Topsham, ME, USA) was used to determine the percentage of cells in the various phases of the cell cycle. Briefly, 1×105 U87 cells were treated with taraxerol acetate (0, 10, 50 and 150 µM) for 48 h. Cells were then collected, washed with ice-cold PBS twice, fixed with 70% alcohol at 4°C for 12 h, and stained with PI in the presence of 3% RNAase A (Sigma-Aldrich) at 37°C for 20 min, prior to analysis with flow cytometry.
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7

Propidium Iodide Cell Cycle Analysis

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Propidium iodide staining solution (PI solution) was used at the following final concentrations: 200 ug/ml RNAase A (Sigma Aldrich, cat# 10109169001), 0.1% Triton-X 100 (v/v) and 20 ug/ml of propidium iodide solution diluted in 1x PBS. HCT116 cells were grown in 6-well plates in a total volume of 2 ml and treated with either test compound or DMSO control for the designated time. After treatment, medium was collected from the cells which were then washed in 1x PBS then removed from the plastic by the addition of in 500 μl Trypsin/EDTA until cells were monodispersed. The trypsin was neutralised by the removed media and the cell suspension was spun at 1000 rpm for 5 minutes. Cells were then washed a further time in ice cold 1xPBS and spun at 1000 rpm for 5 minutes. Cells were then fixed in 4.5ml 70% ice cold ethanol and 0.5 ml ice cold 1xPBS. Cells were left in fixing solution overnight at 4°C until processing. Cells were spun at 1000 rpm for 5 mins and then washed in 1xPBS, re-suspended in 0.5-1 ml of the PI solution at incubated in the dark for 2 hours at room temperature. Cells were then counted and analysed using a Becton Dickinson LSR II cytometer and FCS Express software.
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8

Cell Cycle Analysis of Ovarian Cancer Cells

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Transfected OVCAR3 and OVCA433 cells were harvested and fixed in 70% ethanol at 4°C. The fixed cells were stained with propidium iodide supplemented with RNAase A (Sigma-Aldrich; Merck KGaA) in the dark at room temperature for 30 min. The relative amount of cells in each phase of the cell cycle was analyzed by flow cytometry (FACSCanto II; BD Biosciences) and evaluated using the FlowJo software _v10.8.0 (FlowJo LLC).
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9

Cytotoxicity Assay with Diverse Reagents

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Thiazolyl blue tetrazolium blue (MTT), crystal violet staining solution, Dimethyl sulfoxide (DMSO), RNAase A, Proteinase K, Agarose, and bovine serum albumin were purchased from Sigma-Aldrich. All other chemicals used in this study were obtained from Sigma.
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10

Cell Cycle Analysis by Flow Cytometry

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Asynchronous cells were plated as above and collected as described for the annexin V assay above. Cells were fixed by adding drop-wise 1 mL of ice cold 70% ethanol and stored at −20° C until assay. Cells were pelleted, washed with once with PBS. Cell pellets were re-suspended in L PBS and 200 L extraction buffer (0.2 M Na2HPO4, 0.1M Citrate) followed by the addition of 500 L of propidium iodide (125 Worthington units/mL RNAase A and 50 g/mL propidium iodide, Sigma). The cell suspension was incubated at 37°C for 30 minutes followed by data collection via flow cytometry as above and analysis by FlowJo software using the Dean-Jett-Fox algorithm.
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