Cell cycle distribution was assesses by flow cytometry. Cells were treated with 0.125 g/ml of fig latex or equivalent amount of PBS for 48 hrs. Approximately (1 × 106) cells were harvested from both control and treated flasks. Cells were then washed in PBS and fixed in 70% ice-cold ethanol for 1 hr. Then 500 µl of PI/RNase (Thermofisher, UK) was added to samples and were kept in the dark for 20 minutes at 37 °C. Stained cells were then excited at 488 nm using the FL-3 detector (620 nM) of a BD FACsCalibur flow cytometer (Becton-Dickinson). Acquired data was analysed using CellQuest software (Becton-Dickinson).
Pi rnase
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
PI/RNase is a laboratory equipment product that serves a core function in scientific research and analysis. It is designed to perform specific tasks within a controlled laboratory environment. This product description is presented in a factual and unbiased manner, without extrapolation or interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Click it edu, by Thermo Fisher Scientific (1 mentions)
T75 flask, by Avantor (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Monosodium glutamate, by Merck Group (1 mentions)
Cell culture media, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Nf kb p65, by Cell Signaling Technology (1 mentions)
Pparγ, by Abcam (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Radio immunoprecipitation assay buffer ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg fluorescein isothiocyanate fitc, by Santa Cruz Biotechnology (1 mentions)
3 3 dihexyloxacarbocyanine iodide dioc6, by Thermo Fisher Scientific (1 mentions)
Pparα, by GeneTex (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
6 protocols using pi rnase
1
Cell Cycle Analysis by Flow Cytometry
2
Proliferative Status Analysis via Flow Cytometry
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Cells were grown to 70%−80% confluency in T75 flasks (VWR). They were then treated with serum free media to try to have the cells at a similar point proliferatively. Cells were stained with Click-iT EdU and PI/RNase using established protocols from Thermo Fisher. The fluorescence of both stains was quantified using Flow Cytometry and analyzed using FlowJo (FlowJo, LLC.).
3
Flow Cytometric Analysis of Cell Cycle
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Cell cycle distribution was assessed via flow cytometry. Cells were treated with 100 μg/mL of fig latex or equivalent amount of PBS for 72 h. Approximately (1 × 106) cells were harvested from both control and treated flasks. The cells were then washed in PBS and fixed in 70% ice-cold ethanol for 1 h. Then, 500 µL of PI/RNase (Thermofisher, UK) was added to the samples and they were kept in the dark for 20 min at 37 °C. The stained cells were then excited at 488 nm using the FL-3 detector (620 nM) of a BD FACs Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The acquired data were analysed using CellQuest software (version 5.1) (Becton-Dickinson).
4
Cell Cycle Analysis by Flow Cytometry
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A549 (5 × 105) cells were plated in six-well plates and cultured for 20 h. After vortexing in PBS, the cell samples were fixed with 70% ethanol for 1 h. The cells were isolated by centrifugation and resuspended in PBS. The samples were incubated with propidium iodide (PI)/RNase (Thermo) for 30 min in the dark at room temperature and detected using a FACS Calibur (BD).
5
Cell Cycle Analysis of Hepatocytes and Cardiomyocytes
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RNase-PI (ribonuclease with propidium iodide, Thermo Fisher Scientific, Waltham, MA, USA) was utilized for the analysis of cell cycle of hepatocytes and cardiomyocytes by previously published method of Banerjee A et al. (2020); BD VERSE was utilized for this measurement with excitation at 488 nm and outflow was collected by 585/40 nm band pass filter. Finally the percentages of cells in different phases were analyzed by using the FlowJo software1 (link).
6
Molecular Mechanisms of Monosodium Glutamate-induced Toxicity
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Monosodium glutamate (SRL, India), 5,59-dithio-bis (2-nitro benzoic acid) (DTNB), Triton X-100, Trichloroacetic acid (TCA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Thiobarbituric acid (TBA), Tween 20 and Bovine serum albumin (BSA) were purchased from Sigma Aldrich (USA). Antibodies such as Bcl2, Bax, p21, p53, NF-kB (p65), cleaved caspase 3, cleaved caspase 9 and GAPDH, 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Cell Signalling Technology (USA) and PPAR-α from GeneTex (USA), PPAR-γ from Abcam (USA). Ethidium bromide (EtBr), ethanol, hydrogen peroxide (H2O2), petroleum ether, chloroform, n-hexane, acetone and all other chemicals were procured from Merck (Germany). Anti-rabbit IgG fluorescein isothiocyanate (FITC) and Radioimmunoprecipitation assay buffer (RIPA) buffer were procured from Santa Cruz Biotechnology (USA). 3-3′-dihexyloxacarbocyanine iodide (DiOC6, Molecular Probes), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and RNase-PI were procured from Thermo Fisher Scientific (USA). All the cell culture media, buffer, collagenase type II, Trizol reagent and other reagents were obtained from Gibco (USA) and all other reagents used for this study were of highest quality grade.
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