Cells were harvested by trypzination and fixated in 70 % ice-cold ethanol at −20 °C for a minimum of 24 h. In order to eliminate variation in antibody staining, control as well as treated cells were labelled with different concentrations of Pacific Blue (0.125, 0.031, 0.0062 and 0.00078 ng/μL, respectively) for 30 min at RT in the dark, in accordance with the fluorescent cell barcoding (FCB) technique [28 (link)]. All samples were subsequently mixed in one tube and incubated with primary antibodies (mouse anti-γH2AX (1:500, 05–636, Millipore) and rabbit anti-phospho Histone H3 (ser10) (1:500, CS#9701, Cell Signaling)) diluted in detergent buffer (0.1 % Igepal CA-630, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, 0.5 mM EDTA [pH 7.5]) containing 4 % nonfat milk for 1 h at RT, and afterwards with secondary antibodies; anti-rabbit Alexa488 and anti-mouse Dyelight549 (1:1000, Invitrogen) for 30 min at RT in the dark. Cells were finally incubated with PBS containing Cell Cycle 633/Fx cycle™ far red stain (1 μL/mL,) and PureLink RNAse A (5 μL/mL) (Invitrogen) for 30 min at 4 °C in the dark. Flow cytometric analysis was performed using a LSRII flow cytometer (BD Biosciences) with Diva software, and the four samples were gated based on the Pacific Blue signal before analyzes of cell cycle distribution, γH2AX and phospho Histone H3 (ser10) expression.
Purelink rnase a
Manufactured by Thermo Fisher Scientific
Sourced in United States
PureLink RNase A is a laboratory reagent used for the enzymatic degradation of RNA. It is a purified ribonuclease A enzyme derived from bovine pancreas. The product is designed to effectively remove any remaining RNA from DNA samples, ensuring the purity of the extracted DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Merck Group (5 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (3 mentions)
Attune nxt software v2, by Thermo Fisher Scientific (3 mentions)
Qiaamp dna blood mini kit, by Qiagen (3 mentions)
Ficoll paque centrifugation, by GE Healthcare (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Qiamp dna stool mini kit, by Qiagen (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (2 mentions)
Ebioscience 7 aad viability staining solution, by Thermo Fisher Scientific (2 mentions)
Click it plus edu flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Saponin based permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Ds 11 series, by DeNovix (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
52 protocols using purelink rnase a
1
Multiparametric Flow Cytometry Analysis
2
Reagents and Chemicals Used in Biological Assays
3
DNA Extraction from Leukemic Cells
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Lymphoblasts were purified from bone marrow or peripheral blood specimens by the Ficoll‐Paque centrifugation method according to the manufacturer’s instructions (GE Healthcare). Genomic DNA was extracted from leukemic cells using standard phenol/chloroform‐based methods. Briefly, 1 million cells were lysed in 10 mmol/L Tris‐HCl, 10 mmol/L NaCl, 10 mmol/L EDTA, 20 μg proteinase K, and 0.5% SDS by incubating at 37°C for 16 hours. Total RNA was further removed by adding 500 μg PureLink RNase A (Invitrogen) and incubating for 10 minutes at 37°C. An equal volume of phenol‐chloroform‐isopropanol (25:24:1) was added to lysates and mixed by shaking vigorously, followed by centrifugation at 16 100 g at 4°C for 5 minutes. The upper aqueous phase was transferred to a fresh tube; genomic DNA was then precipitated by adding 2× volume −80°C 100% ethanol. The DNA pellet was washed with 75% ethanol and rehydrated with Tris‐EDTA buffer. Concentration of DNA was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific).
4
Cell Cycle Analysis by Flow Cytometry
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UM-UC-3 and EJ cells were cultured in SSM or SFM for 7 days. Cells were then collected and washed twice with ice-cold PBS, followed by fixation in 70% ice-cold ethanol overnight at 4°C. Subsequently, after being washed and resuspended in PBS, the cells were treated with 0.1% Triton X-100 (Invitrogen; Thermo Fisher Scientific, Inc.), 20 µg/l propidium iodide (PI; Invitrogen; Thermo Fisher Scientific, Inc.) and 0.2 mg/ml RNase (PureLink RNase A; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark for 20 min at room temperature. Flow cytometry was performed to analyze the DNA content and assess the percentage of cells in different phases of the cell cycle (BD FACSLyric; FlowJo version 10.0.7; Becton-Dickinson and Company).
5
Cell Cycle Analysis of BM- and WJ-MSCs
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For cell cycle analysis, BM- and WJ-MSCs from P2, at a confluency of 80–90% were trypsinized, washed with PBS and fixed with 70% cold ethanol. Cells were treated with 0.5 mg/mL RNase A (PureLink RNase A; Invitrogen) and stained with 50 μg/mL propidium iodide (PI, Invitrogen) in the dark for 30 min at 37 °C. DNA content was determined by flow cytometry and the percentage of cells in different phases of cell cycle was assessed. A minimum of 100,000 events were acquired per sample.
6
Cell Cycle Analysis by Flow Cytometry
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After 48 h of siRNA transfection, the cell was collected and fixed by ice-cold 70% ethanol. The cell was then treated by 100 μg/ml of PureLink™ RNase A (Cat#12091021, Invitrogen™) and stained by 50 μg/ml of Propidium Iodide (Cat#P3566, Invitrogen™). After 15 min of incubation at the room temperature, the cells was analysed by the ZE5 Cell Analyzer (BIO-RAD). The data was analyzed by FCS Express Flow Cytometry Software (De Novo Software).
7
Immunofluorescence Staining Protocol
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Cells were grown on coverslips, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min at room temperature. The coverslips were then treated with blocking buffer (2% bovine serum albumin, 5% glycerol, 0.2% Tween 20, and 0.1% NaN3) for 1 hour at room temperature and incubated with primary antibody for 1 hour in blocking buffer at room temperature or overnight at 4°C. Coverslips were washed in PBS and incubated with secondary antibody and 4′,6-diamidino-2-phenylindole (0.1 μg/ml) in blocking buffer for 1 hour at room temperature, washed with PBS, and mounted with Mowiol 4-88 (Cold Spring Harbor protocols). All antibodies used are listed in table S4.
For pre-extraction before fixation, cells were incubated for 5 min at room temperature in cytoskeleton buffer [CSK; 10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.7% Triton X-100]. The permeabilization step was skipped, and cells were directly immersed in blocking buffer. For RNase A treatment, PureLink RNase A (Invitrogen) was added to the CSK buffer to a final concentration of 300 μg/ml, and cells were incubated for 5 min at room temperature.
For pre-extraction before fixation, cells were incubated for 5 min at room temperature in cytoskeleton buffer [CSK; 10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.7% Triton X-100]. The permeabilization step was skipped, and cells were directly immersed in blocking buffer. For RNase A treatment, PureLink RNase A (Invitrogen) was added to the CSK buffer to a final concentration of 300 μg/ml, and cells were incubated for 5 min at room temperature.
8
Intracellular ZBP1 Staining and Detection
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For intracellular ZBP1 staining, cells were fixed and permeabilised in Cytofix/Cytoperm (BD biosciences) according to manufacturer's instructions. When recombinant ZBP1 was used (Fig 6 A), cells were incubated with 0.3 μg ZBP1‐3xFLAG for 1 h in Perm/Wash buffer. In some instances, cells were pre‐incubated for 30 min at 37°C with 30 U Purelink RNase A (Invitrogen) or Turbo DNase I (Ambion). Anti‐FLAG‐APC (Abcam, clone M2) was used to detect ZBP1‐FLAG. Samples were analysed in FACS buffer (PBS, 2 mM EDTA, 1% FCS). 1 μg/ml DAPI (Sigma‐Aldrich) or Live/Dead Fixable Violet (Molecular Probes) was used to exclude dead cells. Data were acquired on Beckmann Coulter CyAn or BD Biosciences LSRFortessa flow cytometers. FCS files were analysed using FlowJo V.10.0.8. For fluorescence microscopy, polyclonal ZBP1‐expressing NIH3T3 cells were seeded on poly‐d ‐lysine (Sigma‐Aldrich)‐coated coverslips (Lab‐Tek). Cells were fixed using 4% paraformaldehyde (ThermoFisher) and blocked and permeabilised for 1 h in PBS + 5% BSA (Sigma‐Aldrich) + 0.1% saponin (Sigma‐Aldrich). Primary antibody (anti‐FLAG‐HRP, clone M2, Sigma‐Aldrich) and detection antibodies (anti‐HRP‐Alexa Fluor 594, Jackson Immunoresearch) were incubated for 1 h in PBS + 5% BSA + 0.1% saponin (Sigma‐Aldrich). Images were acquired using a Zeiss Axiovert S100 inverted fluorescence microscope.
9
Fecal DNA Extraction and Purification
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Total nucleic acids were extracted from frozen fecal samples using the PowerMicrobiome™ RNA Isolation Kit (Qiagen) according to the manufacturer’s instructions and eluted in 108 µl elution buffer. An aliquot of 50 µl was incubated with 10 ng PureLink RNase A (Invitrogen) at 37 °C for 30 min. Subsequently, 5.1 µl sodium acetate (3 M, pH 6.8) and 102 µl isopropanol were added. After 10-min incubation on ice, the DNA was pelleted and washed in ethanol (70% v/v), centrifuged, and dried at room temperature. Finally, purified DNA was re-dissolved in 50 µl RNase-free water and stored at −80 °C.
10
Cell Cycle Analysis of WJ-MSCs
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The cell cycle was evaluated using flow cytometry. WJ-MSCs at a 70–80% confluence were incubated for 24 h, with INFγ + TNFα at a concentration of 5 ng/mL, or else INFγ and TNFα at a concentration of 12.5 ng/mL, or IL-1β at a concentration of 50 ng/mL. Next WJ-MSCs were detached by accutase, washed with PBS and fixed with 70% cold ethanol. The cells were then centrifuged, rinsed with PBS, and resuspended in a staining solution containing 100 µg/mL of RNase A (PureLink RNase A; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 0.1% of Triton X-100 (Sigma Aldrich, Saint Louis, MO, USA) and 10 µg/mL of propidium iodide (PI, Invitrogen). Cells were incubated in the dark for 30 min at RT. DNA content was determined by flow cytometry (BD FACSCanto II) and the percentage of cells in different phases of the cell cycle was assessed. A minimum of 104 events per sample were acquired.
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