To perform the cell cycle analysis, 35 × 103 the MCF7 cells/well and 5 × 104 MCF10A cells/well were seeded in 12-well plates and transfected as described above. 72 h after transfection, medium was collected and adherent cells were harvested by trypsin-EDTA solution to produce a single cell suspension. Then, cells were pelleted by centrifugation, fixed in 70% ice cold ethanol and stored at −20 °C. When ethanol was removed, cells were processed using the PI/RNAse Solution Kit (Immunostep, Salamanca, Spain) for 20 min. The stained cells were analyzed by FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) using FlowJo software v10.2 (Treestar, Ashland, OR, USA), determining the phase of the predominant cell cycle. The experiments were done in duplicate.
Pi rnase solution kit
Manufactured by Immunostep
Sourced in Spain
The PI/RNase solution kit is a laboratory product that provides a solution for the purification and analysis of DNA and RNA samples. It includes reagents for the extraction, separation, and detection of nucleic acids. The kit is designed to facilitate common molecular biology techniques, such as PCR and gel electrophoresis, by ensuring the integrity and purity of the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Gallios cytometer, by Beckman Coulter (2 mentions)
Facscan, by BD (1 mentions)
Agarose, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Mouse antibody against β actin, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Epinephrine, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
7 protocols using pi rnase solution kit
1
Cell Cycle Analysis of MCF7 and MCF10A Cells
2
Cell Cycle Analysis of Compound Treatment
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The compound's effect on cell cycle was investigated by PI/RNase solution kit (Immunostep, Salamanca, Spain). Cells (1×106 cells/well) were incubated with vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC50. After 24 h of incubation, cells were harvested and cell cycle analysis was assessed according to the kit protocol. Briefly, cells were harvested, washed with PBS and centrifuged for 5 min at 300 g. The supernatant was removed and cells were fixed with 70% ethanol for 30 min at 4°. Cells were washed once with PBS supplemented with 2% bovine albumin, centrifuged for 5 min at 300 g and then the supernatant was removed. To the cell pellet it was added 500 µl of PI/RNase solution followed by 15 min incubation at room temperature. Finally, the analysis was performed by flow cytometry as previously described.
3
Evaluating Cytotoxicity and Oxidative Stress
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Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS) and antibiotics were purchased from Gibco (USA). Albendazole (PubChem CID: 2082), methotrexate, calf thymus DNA (CT-DNA), agarose, DMSO, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-acetyl-l -cysteine (NAC), 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), ethidium bromide (EtdBr), protease inhibitor cocktail, hydrogen peroxide, trichloroacetic acid, thiobarbituric acid, glutathione oxidized form (GSSG), NADPH and epinephrine purchased from Sigma Aldrich (Brazil). The phosphatase inhibitor cocktail was from Calbiochem (Merck Biosciences). Antibodies against γH2AX, p53, Bax and Bcl-xL were obtained from Santa Cruz Biotechnology, Inc. (USA). Mouse antibody against β-actin was from Millipore (USA). The secondary antibodies and the kit for chemiluminescence detection of HRP-coupled antibodies were from Millipore (USA). PI/RNAse solution kit from Immunostep (Salamanca, Spain).
4
Assessing Apoptosis and Cell Cycle in Ehrlich Tumor Cells
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Tumor cells in the ascitic fluid (25 μl) were stained using a solution (5 μl) of acridine orange (200 μM) and ethidium bromide (250 μM). Samples were mixed with the staining just prior to microscopy. Cells (300) were visualized and categorized on microscope slides. Live cells appear uniformly green. Early apoptotic cells stain green and contain bright green dots in the nuclei. Late apoptotic cells also incorporate ethidium bromide and therefore stain orange, but, in contrast to necrotic cells, the late apoptotic cells show condensed and often fragmented nuclei [33] .
Cell cycle arrest was evaluated using a PI/RNAse solution kit from Immunostep (Salamanca, Spain) using the procedure suggested by the manufacturer. Ehrlich tumor cells (5×105) were carefully washed with PBS, pelleted and fixed by rapid submersion in ice-cold ethanol (70%) with vigorous vortexing. After overnight fixation at −20 °C the cells were washed with PBS, pelleted, resuspended and incubated (15 min, under room temperature) with PI/RNAse solution. Finally, cells were evaluated by the FACSCanto II (BD Biosciences) cytometer. Data were processed using the Flowing Software 2.5.1.
Cell cycle arrest was evaluated using a PI/RNAse solution kit from Immunostep (Salamanca, Spain) using the procedure suggested by the manufacturer. Ehrlich tumor cells (5×105) were carefully washed with PBS, pelleted and fixed by rapid submersion in ice-cold ethanol (70%) with vigorous vortexing. After overnight fixation at −20 °C the cells were washed with PBS, pelleted, resuspended and incubated (15 min, under room temperature) with PI/RNAse solution. Finally, cells were evaluated by the FACSCanto II (BD Biosciences) cytometer. Data were processed using the Flowing Software 2.5.1.
5
Cell Cycle Analysis of ATDC-5 Cells
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The influence of OE on the ATDC-5 cell cycle was studied using the PI/RNase solution kit (Immunostep, Salamanca, Spain). Briefly, cells (5.7 × 104 cells/cm2) were cultured in 24-well plates and treated with the subcytotoxic concentration of OE. After that, cells were fixed in 70% ice-cold ethanol and kept at 4 °C for 48 h. Samples were then centrifuged, washed in PBS, and stained with a propidium iodide (PI) solution (50 μg/mL) that contained 100 μg/L of RNase A. By using a Beckman Coulter Gallios cytometer (Brea, CA, USA), the DNA content of the PI-stained cells was determined by flow cytometry. At least three separate tests were carried out in triplicate.
6
Cell Cycle Analysis by Flow Cytometry
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The cells were seeded in 6-well plates at a density of 30 × 104 cells/well. After 24 h, the culture medium was removed, and a serum-free culture medium was added to arrest the cell cycle. Then, the culture medium was replaced by DMEM with serum, including extract concentrations equivalent to the IC25 and IC50. After 48 h of incubation, the trypsinized cells were fixed with 70% ethanol in agitation at 4 °C (1 h) and washed twice with PBS. Finally, cells were processed using the PI/RNASE Solution Kit (Immunostep, Salamanca, Spain) to quantify the total content of cellular DNA by FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) using FlowJo software (Treestar, Ashland, OR, USA), determining the phase of the predominant cell cycle.
7
Onion's Impact on Caco-2 Cell Cycle
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The onion’s effect on Caco-2 cell cycle was studied by PI/RNase solution kit (Immunostep, Salamanca, Spain). Briefly, cells were fixed in 70% ice-cold ethanol and stored at 4 °C for 48 h. After centrifugation (413× g, 5 min), cells were rehydrated in PBS and stained with propidium iodide (PI) solution (50 μg/mL) containing RNase A (100 μg/mL). PI-stained cells were analyzed for DNA content by flow cytometry using a Beckman Coulter Gallios cytometer (Brea, CA, USA).
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