Cells were seeded in 6-well plates and grown overnight to 70% confluency. Cells were treated with inhibitors accordingly for 24 h. After 24 h, cells were trypsinized and pelleted. The cells were washed twice in 1 mL PBS. The cells were then fixed in 70% ethanol for 30 min at 4 °C. After fixation, cells were washed with 9 mLs of 1% FBS-PBS and washed again in 10 mLs of 1% FBS-PBS. A mL of master mix containing 10 μg/mL RNaseA (Qiagen) and 50 μg/mL propidium iodide (Sigma–Aldrich, St. Louis, MO) in 1% FBS-PBS was added to the samples followed by an hour incubation at 37 °C in the dark. Acquisition data were acquired and finalized at 10,000 events using the BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) running BD FACSDiva software. Cell cycle analysis was modeled using Modfit version 3.2 (Verity Software House, Topsham, ME, USA).
Modfit version 3
Manufactured by Verity Software House
Sourced in United States
ModFit Version 3.0 is a software application designed for flow cytometry data analysis. The software provides tools for cell cycle analysis, cell population identification, and statistical calculations based on flow cytometry measurements.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Pi rnase staining buffer, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Rnase a, by Qiagen (1 mentions)
Facsdiva software, by BD (1 mentions)
Cell cycle staining kit, by Liankebio (1 mentions)
Fitc conjugated goat anti rabbit antibody, by ZSGB-BIO (1 mentions)
Version 7, by FlowJo (1 mentions)
Triton x 100, by Avantor (1 mentions)
Pi rnase, by BD (1 mentions)
15 protocols using modfit version 3
1
Cell Cycle Analysis by Flow Cytometry
2
Comparative Analysis of Cell Lines
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All values were expressed as mean ± standard deviation (SD). Each value is the mean of at least three independent experiments in each group. One-way analysis of variance (ANOVA) and Tukey–Kramer multiple comparisons test was performed to identify differences among measurements of the groups studied. Graphics were obtained by Prism version 5.0 (CEO and Founder, La Jolla, CA, USA) and ModFit version 3.2 (Verity Software House, Topsham, ME, Estados Unidos) software. P-values <0.05, <0.01, and <0.001 are statistically significant.
3
Cell Cycle Regulation by CEP131
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A549 and SPC-A-1 cells were seeded onto 6-cm dishes at 30–50% confluence, and subsequently transfected with CEP131-siRNA or NC-siRNA. Cells were harvested 48 h following transfection and stained using the Cell Cycle Staining kit (cat. no. WLA010; Wanleibio Co., Ltd.), according to the manufacturer's protocol. DNA content was determined by flow cytometry (BD Biosciences). Cell cycle analysis was modeled using Modfit version 3.2 (Verity Software House, Inc.). Each experiment was repeated three times. The results are presented as the percentage of the total cell count in different phases of the cell cycle, namely G1, S and G2.
4
Cell Cycle Analysis of CB1954 Cytotoxicity
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The three cell lines were plated at a density of 1.0×106 cells/well in a 6-well plate. Following incubation with CB1954 at various concentrations (0, 12.5, 25, 37.5 and 50 µmol/l) for 36 h, the cells were collected, washed twice with ice-cold PBS (pH 7.4), fixed with 70% alcohol for a minimum of 18 h and then stained with PI (50 µg/ml) in the presence of 20 µg/ml RNAse A for a minimum of 30 min prior to flow cytometric analysis. The data were analyzed with ModFit version 3.2 (Verity Software House, Inc., Topsham, ME, USA) and CellQuest version 7.5.3 (BD Biosciences, Franklin Lakes, NJ, USA).
5
Cell Cycle Analysis via Flow Cytometry
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The cell cycle assay kit (cat. no. C1052) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The cells were fixed with 70% ethanol at 4°C for 24 h, and then resuspended in cold phosphate-buffered saline (PBS). Then, the cells were resuspended with a staining solution containing RNase A and propidium iodide (PI), and the DNA content was assessed using a flow cytometer. The number of cells in the different phases was counted using ModFit version 3.2 (Verity Software House, Topsham, ME, USA). Three independent experiments were performed.
6
Cell Cycle Analysis by Flow Cytometry
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PDAC cells were resuspended in 250 μL of phosphate-buffered saline and combined with 750 μL of 100% absolute ethyl alcohol for fixation and then stored for 4 hours at −20°C. Subsequently, the cells were incubated with 500 μL of propidium iodide reagent for 15 min in the dark. The proportion of cells at different stages was then measured by flow cytometry (ModFit version 3.0; Verity Software House, Topsham, ME, USA).
7
Cell Cycle Analysis by Flow Cytometry
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Stably transfected cells were washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol and stained with propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were then sorted using a FACSCalibur instrument (BD Biosciences). Data were analyzed by ModFit, version 3.0 software (Verity Software House, Topsham, ME, USA).
8
Cell Cycle Analysis after AZD1775 and Cisplatin
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After exposure to AZD1775 with/without cisplatin for 24 h, cells were collected, washed with PBS, and fixed in 70% immediately prepared precooled ethanol overnight at 4°C. After washing with PBS three times, cells were stained with propidium iodide (PI)/RNase solutions using a commercial cell cycle detection kit (BD Biosciences) at room temperature for 15 min in the dark according to the instructions, followed by flow cytometry analysis within 1 h (BD Biosciences). Cell cycle distributions were assessed with ModFit version 3.0 software (Verity Software House, Topsham, ME).
9
Cell Cycle Analysis by Flow Cytometry
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Cells were stained using Cell Cycle Staining kit (Lianke Bio, Hangzhou, China) and analyzed by flow cytometry. In brief, a pellet of ~2×105 cells was formed by centrifugation and the cells were washed with PBS. Subsequently, 1 ml cold 75% ethanol was added to the cells at −20°C overnight. Subsequently, the cells were rehydrated with PBS for 15 min and incubated with 1 ml DNA staining solution at room temperature. The cells were examined using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The results were analyzed using the ModFit version 3.0 software (Verity Software House, Topsham, ME, USA).
10
Apoptosis and Cell Cycle Analysis
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Cells were treated with 0.3 μM AZD1775 in the presence or absence of 20 μM olaparib. For apoptosis analysis, cells were collected, washed in PBS and double-stained using an Annexin V-Phycoerythrin (PE)/7-amino-actinomycin (7-AAD) apoptosis detection kit (BD Biosciences, Erembodegem, Belgium) following the vendor’s protocol. For cell cycle analysis, cells were digested with trypsin, washed in PBS, fixed in 70% immediately prepared precooled ethanol overnight at 4 °C. After PBS washing, cells were stained with a propidium iodide (PI)/RNase staining buffer (BD Biosciences) at room temperature for 15 min in the dark according to the manufacturer’s instruction. For quantifying pHH3 and γH2AX positive cells at separate cell cycle stages, cells were fixed in 70% ice-cold ethanol overnight at 4 °C, permeabilized with 0.5% Triton X-100 (Amresco, Solon, OH) for 10 min at room temperature, incubated with primary antibodies against pHH3 (1:300) and γH2AX (1:200), probed with the FITC-conjugated goat anti-rabbit antibody (1:200; ZSGB-BIO, Beijing, China) and stained with a PI/RNase staining buffer (BD Biosciences) as described in protocols. All steps were followed by PBS washing. Samples were detected by flow cytometry within 1 h (BD Biosciences) and analyzed using FlowJo Version 7.6.1 software (FlowJo, Ashland, OR) or ModFit Version 3.0 software (Verity Software House, Topsham, ME).
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