Cell cycle analysis was performed by measuring DNA content to distinguish between different phases of the cell cycles. Fluorescence intensity, which directly correlated with the amount of DNA contained in a cell, was measured. Concurrent parameter measurements made it possible to discriminate between S, G2, and mitotic cells. As the DNA content doubles during the S phase, the intensity of the fluorescence increases, making it possible to ascertain the effect of U. parvum infection on cell proliferation. Cell cycle analysis was performed using the flow cytometer, as described previously using the Coulter DNA Prep Reagents Kit (Beckman Coulter, Indianapolis, IN) (Tantengco et al., 2021b (link)). Briefly, cells were harvested by trypsinization and centrifuged for 5 min at 3000 × g. Cell pellets were washed with cold 1× PBS and centrifuged at 3000 × g for 5 min. Cell pellets were fixed with 500 μL 70% ethanol for 15 min. Cell pellets were washed with cold 1× PBS and centrifuged at 3000 × g for 5 min. Then, 500 μL of the prep stain was added to the tubes, vortexed, and run immediately on the CytoFlex flow cytometer (Beckman Coulter). After selecting for single cells, gating was set for the control cells and applied to histograms for each of the treatments in different cervical cells using CytExpert (Beckman Coulter).
Coulter dna prep reagents kit
Manufactured by Beckman Coulter
Sourced in United States, France
The Coulter DNA Prep Reagents Kit is a laboratory product designed for the preparation of DNA samples. It contains a set of reagents necessary for the extraction and purification of DNA from various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex flow cytometer, by Beckman Coulter (4 mentions)
Cytexpert, by Beckman Coulter (3 mentions)
Modfit lt 2, by Verity Software House (3 mentions)
Trypsin edta, by Corning (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Kaluza analysis software 2, by Beckman Coulter (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
8 mm pore transwell inserts, by Corning (1 mentions)
Tgf β1, by Corning (1 mentions)
Tgf β1, by Beyotime (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Cell counting kit 8 cck 8 assay, by Beyotime (1 mentions)
Apoptosis detection kit, by BestBio (1 mentions)
Fc500 flow cytometry system, by Beckman Coulter (1 mentions)
Coulter epics xl mcl flow cytometer, by Beckman Coulter (1 mentions)
Fc500 flow cytometry system with cxp software, by Beckman Coulter (1 mentions)
Multicycle for windows, by Beckman Coulter (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
22 protocols using coulter dna prep reagents kit
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis of SiRNA-Treated CAL 27
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SiRNA-treated CAL 27 cells were collected and prepared by using a COULTER DNA PREP Reagents Kit (Beckman-Coulter, USA) according to the manufacturer’s instruction. Cell cycle analyses were performed using a BD Biosciences FACSCalibur flow cytometer and Modfit LT software (Verity software house).
3
Cell Cycle Progression Analysis
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Cell cycle progression was determined by flow cytometry using the Cytoflex instrument purchased from Beckman Coulter (Milan, Italy). To this aim, cells were stained using Coulter DNA PREP Reagents Kit, (Beckman Coulter) according to manufacturer's instructions. Briefly, TPC-1 and CAL62 (5 × 104 cells) were treated with AFPE for 24 hours at the doses of 250 and 500 μM at 37°C with 5% CO2. After cellular detachment, cells were incubated with DNA PREP LPR reagent for 15 minutes and then with DNA PREP STAIN, containing 50 μg/ml of propidium iodide, for 1 hour. TPC-1 and CAL62 stained cells were acquired using the Cytoflex flow cytometer (Beckman Coulter). Cell cycle progression was analyzed using Kaluza Analysis Software 2.1 (Beckman Coulter) applying the Michael Fox algorithm.
4
Profiling TGFβ1-induced Senescence in AML Cells
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AML cell lines KG-1a was ordered from the Shanghai cell bank of the Chinese Academy of Sciences and cultured in RPMI 1640 (Gibco) supplemented with 20% fetal bovine serum. Cells were maintained at 37 °C in a 5% CO2 incubator. KG-1a was precultured with 10 ng/ml TGFβ1 (PeproTech) for 48 h and used for Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, China), apoptosis detection (Apoptosis Detection Kit, BestBio), cell cycle detection (COULTER DNA PREP Reagents Kit, Beckman). Cell cycle phase distributions were generated by fitting DNA content histograms to applicable models using ModFit LT software for Windows (Version 5.0). KG-1a cells were stained with Senescence‐associated‐β‐Galactosidase (SA‐β‐Gal) staining (Beyotime) according to the instructions after TGFβ1 treatment for 7 days, and cells that stained green‐blue were evaluated as positive senescent cells. Cell suspension (1 × 105, 200 μl of serum-free medium) after TGFβ1 pretreatment was seeded onto 8-mm Pore Transwell Inserts (Corning) in 24-well plates with or without matrigel (BD biosciences) and photographed within 24 h.
5
Cell Cycle Analysis of DAPT Exposure
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Cell cycle analysis was performed using the Coulter DNA Prep™ Reagents kit (Beckman Coulter). Cells were prepared as previously described. The cells were then exposed to various concentrations of DAPT (1, 2, 5 and 10 μM) for 48 h at 37°C. Cells were harvested, washed with cold PBS, fixed with 70% ethanol and stored at 4°C for subsequent cell cycle analysis. For detecting DNA content, cells were incubated in the dark at room temperature with 0.5 ml RNase A for 20 min and with 1 ml PI for 20 min. The DNA content of the cells was measured using the Beckman Coulter FC500 Flow Cytometry system. The percentage of cells in G1, S and G2/M phases was calculated.
6
Analyzing Cell Cycle of MCF-7 Cells Exposed to DMSA-SPION
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MCF-7 cells were plated in 25-cm2 flasks and incubated with DMSA-SPION for 24 h. Analysis of cell cycle was performed by flow cytometry using propidium iodide (PI) labeling of DNA cell content. Cells were trypsinized (also harvesting possible detached cells) and centrifuged at 1200 rpm for 5 min. After centrifugation, pellet was resuspended in 100 μl of culture medium without phenol red. Then, it was added 50 μl of Coulter DNA Prep Reagents Kit (Beckman-Coulter Inc, California, USA), 1 ml of PI solution with RNase and incubated for 30 min at 37°C. Both reagents were purchased from Sigma-Aldrich. Distribution of cells in different phases of cell cycle was determined using a Coulter Epics XL-MCL flow cytometer (Beckman-Coulter Inc.) with an argon laser line (488 nm), complemented with appropriate filters, and a minimum of 104 labeled cells per sample were analyzed in each experimental condition. Percent of cells in each phase of the cell cycle was compared with that of control cells (without nanoparticles incubation). At least 10000 fluorescent events were counted per sample.
7
Flow Cytometric Analysis of Adenosine-Treated HTR-8/SVneo Cells
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HTR-8/SVneo cells treated with or without adenosine were detached using trypsin, fixed with 500 μL iced 70 % ethanol, and stained using a Coulter DNA-Prep Reagents Kit (Beckman Coulter, USA). The stained HTR-8/SVneo cells were then analyzed using a CytoFlex Flow Cytometer (Beckman Coulter, USA). All experiments were repeated independently three times.
8
Aminoquinol's Effect on Cell Cycle
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Cells (4×104) were seeded in 24-well plates in DMEM medium containing 0.125% FBS for 24 h, and then treated with DMEM medium containing 10% FBS and various dose of aminoquinol for indicated time at 37°C. At the end of experiments, cells were fixed in ice-cold 70% ethanol and stained using a Coulter DNA-Prep Reagents kit (Beckman Coulter, Brea, CA, United States). Cellular DNA content of 1×104 cells from each sample was determined using an EPICS xL4 flow cytometer (Beckman Coulter). The cell cycle phase distribution was analyzed using ModFit LT 2.0 software (Verity Software House, Topsham, ME, United States). All data were obtained from two separate experiments of which each was performed in triplicate.
9
Cell Cycle Analysis of Treated Cells
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Cell cycle analysis was performed using a commercial kit (Coulter DNA Prep™ Reagents Kit; Beckman Coulter, Fullerton, CA, USA). Cells were plated in six-well plates and incubated overnight before treatment (Mock, 50 nmol/l dsCon and 50 nmol/l dsPAWR-435). Following treatment, cells were harvested, then washed twice with pre-chilled PBS and resuspended in 100 μl PBS at a concentration of 1×106 cells/ml. Each cell sample was mixed with 100 μl DNA Prep LPR (contained in Coulter DNA Prep™ Reagents Kit), gently mixed by vortex and incubated in the dark at room temperature (25°C) for 20 minutes. Then each was mixed with 1 ml of stain (DNA Prep Stain; contained in Coulter DNA Prep™ Reagents Kit), gently mixed by vortex and again incubated in the dark at room temperature (25°C) for 20 minutes. Finally, cell cycle analysis was performed within 1 hour using flow cytometry (Beckman Coulter FC500 Flow Cytometry System with CXP Software; Beckman Coulter, Fullerton, CA, USA), and the raw data was analyzed by Multicycle for Windows (Beckman Coulter).
10
Cell Cycle Analysis of CSE-Treated AECs
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CSE treated and control AECs were harvested after media collection using trypsin EDTA (Corning, Corning, NY) and centrifuged for 10 minutes at 3000 RPM. The supernatant was removed and cells were resuspended in 50 μL PBS. Cell cycle analysis was performed using the Coulter DNA Prep Reagents Kit (Beckman Coulter, Indianapolis, IN). Briefly, 50 μL of DNA Prep LPR was added to each sample and vortexed. Then 1.0 mL DNA Prep Stain was added to the tubes, vortexed and run immediately on the Cytoflex flow cytometer (Beckman Coulter). After selecting for single cells, gating was set for the control cells and applied to histograms for the CSE treated AECs using Cytexpert (Beckman Coulter).
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