2 × 106 of HCT116 cells were inoculated in 60 mm Petri dishes for 24 h and then treated with compound 7f at its IC50 concentration for 48 h. In this study, non-treated HCT116 cells were used as a negative control. After the incubation period of 48 h, HCT116 treated cells were centrifuged at 1,200 rpm at 4 °C for 10 min. After disposing of the supernatant, the cell pellet was suspended in phosphate-buffered saline (PBS) and then centrifuged at 1200 rpm for 10 min. 70% cold ethanol was added overnight to allow the fixation of the cell pellet. After the step of centrifugation, the propidium iodide (PI) mixture was added to the cell pellet for 30 min at room temperature in the darkness. Subsequently, cells were subjected to Epics XL-MCL flow cytometer (BeckmanCoulter, Miami, FL) for analysis of DNA content88 . The percentage of cells in different phases of the cell cycle was analyzed by Multicycle software (Phoenix Flow Systems, San Diego, CA).
Multicycle software
Manufactured by Phoenix Pharmaceuticals
Sourced in United States, Japan
Multicycle software is a laboratory equipment product that provides a platform for the management and analysis of experimental data. It serves as a tool for organizing and processing information obtained during various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (9 mentions)
Propidium iodide, by Merck Group (6 mentions)
Fc500 flow cytometer, by Beckman Coulter (5 mentions)
Facscan flow cytometer, by BD (4 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
Epics xl mcl flow cytometer, by Beckman Coulter (3 mentions)
Rnase a, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Epics flow cytometer, by Beckman Coulter (2 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (2 mentions)
Test plus dna reagent kit, by BD (2 mentions)
Ribonuclease a, by Merck Group (2 mentions)
Accuri c6, by BD (2 mentions)
Facs aria fusion cytometer, by BD (2 mentions)
Cxp software, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Epics profile 2 flow cytometer, by Beckman Coulter (2 mentions)
Annexin 5, by BD (2 mentions)
Apoptotic detection kit, by BD (2 mentions)
Facscan flow cytometer, by Beckman Coulter (2 mentions)
89 protocols using multicycle software
1
Cell Cycle Analysis of HCT116 Cells
2
Cell Cycle and Apoptosis Quantification
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The effects of RuPOP and/or TRAIL on the cell cycle progression and the induction of apoptotic cell death were quantified by flow cytometric analysis. Briefly, treated or untreated cells were trypsinized, washed with PBS and fixed with 70% ethanol overnight at −20°C. The fixed cells were washed with PBS and incubated with a PI working solution for 4 h in darkness. The stained cells were analyzed with flow cytometer (Beckman Coulter, Fullerton, CA). Cell cycle distribution was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA). The proportion of cells in G0/G1, S, and G2/M phases was represented as DNA histograms. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. For each experiment, over 10000 events per sample were recorded.
Caspase activity was detected by fluorescence assay using specific substrates. Briefly, cell lysates were placed in 96-well plates and then the specific caspase substrates were added. Plates were incubated at 37°C for 2 h and caspase activity was determined by measuring the fluorescence intensity on a microplate spectrophotometer (Spectra Max M5, Bio-Tek) with the excitation and emission wavelengths set at 380 and 440 nm, respectively.
Caspase activity was detected by fluorescence assay using specific substrates. Briefly, cell lysates were placed in 96-well plates and then the specific caspase substrates were added. Plates were incubated at 37°C for 2 h and caspase activity was determined by measuring the fluorescence intensity on a microplate spectrophotometer (Spectra Max M5, Bio-Tek) with the excitation and emission wavelengths set at 380 and 440 nm, respectively.
3
Measuring Cellular Oxidative Stress
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Cells were treated with anticancer agents for 48 h. Cells were then labelled with 5 µmol/L CM-H2DCF-DA to measure ROS levels by flow cytometry using the Multicycle software (Phoenix, San Diego, CA, USA).19 (link)–21 (link) The relative ROS level was measured and normalised by the level determined in untreated counterpart cells, set as 1 (X).
4
Cell Cycle Analysis Protocol
5
Cell Cycle Analysis of Neural Progenitors
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HH14 embryos were recovered 16 or 20 h after coelectroporation with the pSox2:EGFP and pTis21:RFP reporters, in the absence (for PCR analysis) or additional presence (for cell cycle analysis) of control or sh-S1/5 vectors. Cell suspensions were obtained from pools of six to eight dissected neural tubes after digestion with trypsin-EDTA (Sigma-Aldrich) for 10–15 min and further processed on a cell sorter (FACS Aria III; BD) for EGFP and RFP fluorescence. To analyze the DNA content, the samples were incubated with 10 µg/ml of Hoechst (Sigma-Aldrich) at 37°C for 30 min. The cellular DNA content was analyzed in single fluorescence histograms using the Multicycle software (Phoenix Flow Systems). The mean ± SEM represents the percentages of cells in G1, S, and G2–M from the analysis of 9–10 cell pools per experimental condition. At least 5,000 cells for each progenitor population (PP, PN, and NN) were analyzed per pool. Alternatively, dissociated cell pools were processed for pH3 immunocytochemistry and analyzed for EGFP, RFP, and Cy5 fluorescence. The data are presented as the means ± SEM obtained from four to five cell pools per experimental condition.
6
Cell Cycle Analysis of PDAC Cells
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PDAC cells were treated with the indicated drugs for up to 48 h. DNA content was determined by propidium iodide (PI) staining and flow cytometry analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), as previously described [34 (link)]. Cell cycle analysis was performed using Multicycle software (Phoenix Flow Systems, Inc., San Diego, CA, USA). Cell death is expressed as the percent of cells with sub-G1 DNA content. Histograms were created using FlowJo v7.6.5 (Tree Star, Ashland, OR, USA). The extent and direction of antileukemic interaction for VS-5584 and BVD-523 was determined by calculating the combination index (CI) values using CompuSyn software (Combosyn Inc., Paramus, NJ). CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively [31 (link), 35 (link)].
7
Cell Proliferation and Cell Cycle Analysis
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Cell proliferation was evaluated by using the CellTiter-Glo cell viability assay (Promega) following the manufacturer’s instructions. Briefly, 1500 cells/well were plated in 96-well plates in the presence of miRNAs, siRNA, or inhibitors. The plates were then incubated for 3–7 days, then 100 μL of CellTiter-Glo reagent was added to lyse the cells. After a 10-min incubation at room temperature, luminescence was recorded in a luminometer with an integration time of 1 s per well.
For cell cycle analysis, transfected cells were harvested at 48 h and fixed at 4 °C. Propidium iodide (50 μg/mL) was used to stain the fixed cells. DNA content was analyzed using a FACSCalibur instrument (Becton Dickinson Bioscience). Cell cycle fractions were quantified with Multicycle Software (Phoenix Flow Systems) and analyzed by FlowJo software.
For cell cycle analysis, transfected cells were harvested at 48 h and fixed at 4 °C. Propidium iodide (50 μg/mL) was used to stain the fixed cells. DNA content was analyzed using a FACSCalibur instrument (Becton Dickinson Bioscience). Cell cycle fractions were quantified with Multicycle Software (Phoenix Flow Systems) and analyzed by FlowJo software.
8
Cell Cycle and Apoptosis Analysis
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Cell fate of cell cycle and apoptosis in CS-1 and SW1353 cells was measured by flow cytometry. With respect to the evaluation of cell cycle, we harvested tumor cells for fixation with 70% ethanol, prior to the administration of RNase A (Thermo Scientific, NY, USA) for 0.5 h. Propidium Iodide (Sigma-Aldrich, MO, USA) was used to stain cells for 0.5 h. Flow cytometry analyses were then deployed to investigate the cell fate and MultiCycle software (Phoenix Flow Systems, CA, USA) was employed to count the cellular events in each cell cycle stage. With respect to the exploration of cell apoptosis, we harvested tumor cells for further staining with FITC annexin V and Propidium Iodide (Invitrogen, NY, USA) for 0.5 h. Flow cytometry was used to analyze the effect of palbociclib on cell apoptosis.
9
Cell Cycle Analysis of Synchronized Cells
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The three groups of cells (Hca-F, Sulf-1-Hca-F and Nc-Hca-F cells) were synchronized at G0/G1 phase by growth in 100% confluence with reduced serum for three days [21 (link)]. The cells were then passaged and cultured for 24hrs after which they were harvested in the log phase of growth, washed twice with ice-cold PBS and fixed in 75% cold ethanol overnight at 4°C. The following day the cells were washed twice with ice-cold PBS after discarding the ethanol, following which 50μg/ml of RNase (Sigma, USA) was added for 30 min and then stained with 20μg/ml of propidium Iodide (Sigma, USA) overnight in darkness. The cells were analyzed by flow cytometry (Beckman Coulter, USA) and the data were analyzed by Multicycle software (Phoenix Flow Systems, San Diego, USA) to get the cell cycle distributions.
10
Cell Cycle Analysis of SK-MES-1 Cells
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SK-MES-1 cells (1.2×106 cells/well) were incubated in 6-well plates at 37°C for 24 h, and the cell culture medium was replaced with fresh medium containing 10% FBS with or without erlotinib at 37°C for another 72 h. The cells were trypsinized, fixed in ice-cold 70% ethanol overnight and stained with propidium iodide containing 1 mg/ml RNase (Sigma-Aldrich; Merck KGaA) according to the instructions of the Cell Cycle Phase Determination kit (Cayman Chemical Company). The samples were analyzed using a flow cytometer (FACSCalibur; BD Bioscience). The cell cycle parameters from 10,000 events were analyzed using multi-cycle software (version 3.1.1, Phoenix Flow Systems).
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