Chemicals used in this study were of analytical grade and were commercially purchased. DNase I, RNase A, and lysozyme were purchased from Bio Basic Canada. Restriction endonucleases, T4 DNA ligase, Antarctic phosphatase, and Phusion DNA polymerase were from New England Biolabs. ε-Rhodomycinone was provided by Dr Kristiina Ylihonko.
Rnase a
Manufactured by Bio Basic
Sourced in Canada
RNase A is a laboratory reagent used for the degradation of RNA. It is an enzyme that specifically cleaves the phosphodiester bonds in single-stranded RNA molecules, resulting in the fragmentation of the RNA.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Dnase 1, by Bio Basic (2 mentions)
Facscalibur cytometer, by BD (2 mentions)
Cellquest pro software, by BD (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Lysozyme, by Bio Basic (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Gfp trap beads, by Proteintech (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Phenol chloroform isoamyl alcohol, by Bioneer (1 mentions)
Monolith nt 115pico instrument, by NanoTemper (1 mentions)
13 protocols using rnase a
1
Microbial Metabolite Synthesis Protocols
2
Cell Cycle Analysis by Flow Cytometry
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After treatment, the harvested cells were detached for the cell cycle analysis with 0.25% trypsin- EDTA (GIBCO, NY, USA), washed with PBS and then fixed with ice-cold 70% ethanol for 2 h. Cells were rewashed with PBS and treated with 50 μg/ml RNase A (Bio Basic Inc., ON, Canada) for 30 min. After that, cells were incubated with Propidium Iodide (PI; Sigma-Aldrich, MO, USA). Flow cytometry analysis was performed using the BD FACSCalibur cytometer.
3
GFP-trap Immunoprecipitation from Transfected 293T Cells
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293T cells were seeded at 6 × 106 cells/100 mm dish and transfected on the following morning with 10 μg of plasmid DNA and Lipofectamine2000 (ThermoFisher) as per the manufacturer's protocol. After 24 h incubation, cells were washed with PBS and 1 mL lysis buffer (1% Triton X-100; 150 mM NaCl; 20 mM Tris–HCl, pH 7.5; 0.1 mg/ml PMSF) and incubated 5 min on ice. Petri dishes were scraped, and protein lysis was completed by a couple up and down. Lysates were sonicated on ice at 25% amplitude, 5 s for four times. Debris were then pelleted at 13 000 RPM, 4°C for 10 min. Lysates were dosed for total protein in triplicate using standard Bradford assay (Thermo Scientific Coomassie Protein Assay). One milligram of lysate was DNAse and RNAse treated using 5 μg of DNAse I (Sigma) and 5 μg of RNAse A (Bio Basic), 10 min at room temperature. GFP-trap beads (Chromotek) were washed twice in lysis buffer; 20 μl were added per IP and IP reactions were completed at 1 mL with lysis buffer. IP was performed for 4 h at 4°C on a tube rotator. Tubes were then spinned at 2000 RPM, the supernatant was removed, and beads were washed three times with lysis buffer and twice with PBS. Immunoprecipitates were subjected to mass spectrometry preparation, or resuspended in 1× Laemmli buffer, boiled, and submitted to western blotting.
4
Apoptosis and Cell Cycle Analysis of CAP-Treated Glioblastoma Cells
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After treating U-87 MG cells with CAPs IC50 doses obtained from the MTT assay, the cells were incubated for 48 h and 72 h in the helium and argon gas plasma groups, respectively. The Annexin V-FITC/PI apoptosis detection kit (Bender MedSystems, Austria) was utilized to quantify early and late apoptosis of U-87 MG cells after CAP treatment, following the manufacturer's instructions. In brief, after treatment, cells were washed in PBS, then resuspended in 100 μl of binding buffer and stained with 5 μl FITC-conjugated Annexin-V for 15 min in darkness at room temperature. Subsequently, samples were washed, resuspended in 250 μl binding buffer, and incubated with 5 μl Propidium Iodide (PI) from Sigma-Aldrich, MO, USA for 10 min. The results were analyzed using the BD FACSCalibur cytometer. Additionally, the DNA content was determined to quantify the G1, S, and G2/M phases of the cell cycle. For this purpose, following treatment, the cells were detached using 0.25% trypsin–EDTA (GIBCO, NY, USA). After detachment, the cells underwent PBS washes, followed by fixation with ice-cold 70% ethanol for 2 h. Subsequently, the cells were washed using PBS, treated with 50 μg/ml RNase A (Bio Basic, Canada) for 30 min, and then incubated with Propidium Iodide (PI; Sigma-Aldrich, USA). Flow cytometry analysis was carried out using the BD FACSCalibur cytometer.
5
Cell Cycle and ROS Analysis Protocol
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For cell cycle analysis, cells were detached and harvested at each time point with trypsin-EDTA (Gibco-BRL), washed with cold PBS, and fixed in 70% cold ethanol for 2 h. Cells were washed in 2% FBS containing cold PBS, resuspended in PBS with 100 μg/mL RNase A (Bio Basic Inc., ON, Canada) for 30 min at 37 °C, and then stained with 10 μg/mL propidium iodide (PI; Sigma-Aldrich) for 15 min at room temperature in the dark.
To measure intracellular ROS levels, cells harvested by trypsin were stained with 5 μM carboxy-H2DCFDA in HBSS for 30 min in a CO2 incubator and washed twice with cold PBS. Flow cytometry was performed using a BD FACSCalibur (BD Biosciences, NJ, USA), analyzed with BD CellQuest Pro software (BD Biosciences), and then data was analyzed using Flowing software (v 2.5.1;http://www.uskonaskel.fi/flowingsoftware ).
To measure intracellular ROS levels, cells harvested by trypsin were stained with 5 μM carboxy-H2DCFDA in HBSS for 30 min in a CO2 incubator and washed twice with cold PBS. Flow cytometry was performed using a BD FACSCalibur (BD Biosciences, NJ, USA), analyzed with BD CellQuest Pro software (BD Biosciences), and then data was analyzed using Flowing software (v 2.5.1;
6
Genomic DNA Extraction from Fungal Mycelium
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For isolation of genomic DNA, 400 μL of Soil DNA Extract buffer (100 mM NaCl, 50 mM EDTA, 0.25 M Tris-HCl, 5% SDS), 400 μL of 2×CTAB buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 1% polyvinyl pyrrolidone), and 500 μL phenol-chloroform-isoamylalcohol (25∶24∶1, Bioneer, Korea) were added to 0.1–0.5 g lyophilized or fresh mycelium and briefly vortexed. After 5 min of incubation at room temperature, samples were centrifuged at 13,000 rpm at 4°C, for 5 min. Supernatants were mixed with 0.7 volumes isopropanol and centrifuged for 10 min at 4°C. After washing with 70% ethanol, air dried samples were eluted in 50–100 μL TE and treated with RNase A (Bio Basic Inc, Canada) for 30 min at 60°C.
7
Microscale Thermophoresis Analysis of RNase Binding
8
Apoptosis Analysis by Flow Cytometry
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After treatment with BTCI, cells were digested with trypsin, harvested, washed twice with ice-cold PBS, fixed with 75% ethanol at −20 °C overnight, washed again and then incubated with RNase A (25 μg/ml, Bio Basic Inc., Markham, Ontario, Canada) at 37 °C for 30 min. Cells were washed once with PBS and incubated with PI (50 μg/ml) for 30 min at room temperature in the dark. The cells were re-suspended in 500 μl PBS and subjected to flow cytometry on a Cytomics FC500 flow cytometer (FACScan; BD Biosciences). A sub G0/G1 population seen to the left of the G0/G1 peak represents DNA fragmentation caused by apoptosis.
9
Cell Cycle Analysis of U2OS and Fibroblasts
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U2OS cells were grown to confluence and then serum starved for 24 hours. Cells were subsequently incubated in fresh media containing 20 % FBS for the indicated times before being harvested for immunoprecipitation and flow cytometry analysis. Primary human fibroblasts were grown to confluence and further cultured for 3 days. The cells were then serum starved for two days, trypsinized and replated in fresh medium before being harvested at the indicated times for immunoprecipitation and flow cytometry analysis. For flow cytometry analysis, the cells were harvested by trypsinization and fixed with 75 % ethanol. Following centrifugation, cells were incubated for 30 min at 37 °C in PBS containing 100 µg/ml RNase A (Biobasic, RB0473) for 30 min before DNA staining with 50 µg/ml propidium iodide. Cell DNA content was determined with a FACSCalibur™ flow cytometer (BD Biosciences®) and analyzed with the CellQuest™ Pro software (BD Biosciences).
10
Cell Cycle Analysis of Transfected U87 and C6 Cells
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U87 and C6 cells (4 × 104 cells/well) were cultured in 24-well plates in triplicates. After 24 h, the cells were transfected with pCL19A or pCDNA3.1( +) mock vectors (1 μg/well) using Lipofectamine3000 reagent (1 μL/well) (Thermo Fisher Scientific, USA). At 72 h, 96 h, and 120 h post-transfection, cells were harvested and fixed in 70% cold ethanol at 4 °C for 24 h. The fixed cells were stained with 500 μL PBS containing 50 μg/mL propidium iodide (Sigma, USA), 100 μg/mL RNase A (BioBasic, Canada), and 0.1% Triton X-100 (Sigma, USA), and incubated for 30 min at room temperature in the dark. All samples were assessed for the cellular DNA content by a FACS Calibur Flow Cytometer (BD biosciences, USA), and the data were analyzed by FlowJo software.
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