After treatment, cells were washed in 1X PBS, fixed in 70% ice-cold ethanol, stained with 10 µg/ml PI (MP Biomedicals), 10 kU/ml RNAse (Sigma-Aldrich; Merck KGaA) and 0.01% Nonidet™ P40 (NP40; Sigma-Aldrich; Merck KGaA) overnight at 4°C in the dark and analyzed by flow cytometry. Data analysis was performed using ModFit 4.1 (DNA Modelling System, Verity Software House, Inc.).
Modfit 4
Manufactured by Verity Software House
Sourced in United States
ModFit 4.1 is a software application for flow cytometry data analysis. It is designed to analyze cell population distributions and model cell cycle phases. The software provides tools for data visualization, gating, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Fxcycle pi rnase staining solution solution, by Thermo Fisher Scientific (2 mentions)
Facs canto 11 system, by BD (2 mentions)
Facsdiva software version 8, by BD (2 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Fcs express 3, by De Novo Software (1 mentions)
Anti brdu primary antibody, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by Verity Software House (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Bryte hs flow cytometer, by Bio-Rad (1 mentions)
Winlist 8, by Verity Software House (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Dojindo Laboratories (1 mentions)
Propidium iodide, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
12 protocols using modfit 4
1
Cell Cycle Analysis by Flow Cytometry
2
BrdU Incorporation for Cell Cycle Analysis
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The incorporation of 5-bromo-2’-deoxyuridine (BrdU) was used to identify actively replicating S-phase cells by two parameter flow cytometry. Cells were incubated with BrdU (30 μM; Sigma-Aldrich Canada Ltd.) for 30 min immediately prior to collection in order to label nascent DNA. Cells were collected and fixed at -20°C in 70% ethanol for a minimum of 1 h. Detection of BrdU incorporated into single- stranded DNA was performed using a primary anti-BrdU antibody (1:100; Phar- Mingen) and secondary anti-mouse FITC-conjugated antibody (1:15; Sigma-Aldrich Canada Ltd.), as described previously [39 (link)]. DNA was stained with propidium iodide (30 μM in PBS) and then samples were analyzed by fluorescence-activated cell sorting using a Becton Dickinson Accuri C6 benchtop flow cytometer and FCS Express 3 software (DeNovo Software). One parameter flow cytometry experiments were performed similarly except that cells were not incubated in BrdU and the fixed cells were simply stained in 30 μM propidium iodide in PBS. Cell cycle distribution in one parameter flow cytometry experiments was determined based on DNA content using Modfit 4.1 (Verity Software House).
3
Cell Cycle Analysis by Flow Cytometry
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Cells were carefully dissociated into single-cell suspensions by trypsin (Gibco), washed twice with PBS, and then fixed overnight with cold 70% ethanol. Fixed cells were washed twice with PBS, followed by ribonuclease (100 μg/ml; Sigma-Aldrich) treatment and PI (50 μg/ml; Sigma-Aldrich) staining for 30 min at 37°C. Approximately 1 × 106 cells were analyzed using FACSCanto II (Becton Dickinson) to determine the cell cycle distribution pattern. The percentages of cells in the G1, S, and G2-M phases of the cell cycle were analyzed using ModFit 4.1 (Verity Software House).
4
Cell Cycle Analysis and Apoptosis Induction
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Cells were seeded in 25 cm2 polystyrene flasks at a density of 520 000 cells per flask. For cell cycle analysis, two different concentrations of afatinib were tested. After 24 h of incubation, cells were collected by trypsinization, fixed with 70% cold ethanol and stored at −20°C. DNA staining was performed with a solution containing RNase (5 μg/ml) and 7-AAD (0.05 μg/μl).
Induction of apoptotic cell death by the different sequences of incubation with afatinib and/or cisplatin was investigated using Annexin V-FITC/Propidium Iodide assay (BD Biosciences).
Analysis was performed using a BD Bioscience FACSCalibur flow cytometer while data were processed and analyzed with ModFit 4.0 (Verity Software House). Three independent experiments were performed.
Induction of apoptotic cell death by the different sequences of incubation with afatinib and/or cisplatin was investigated using Annexin V-FITC/Propidium Iodide assay (BD Biosciences).
Analysis was performed using a BD Bioscience FACSCalibur flow cytometer while data were processed and analyzed with ModFit 4.0 (Verity Software House). Three independent experiments were performed.
5
Annexin V-FITC and PI Apoptosis and Cell Cycle Analysis
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All cells were incubated for 96 h at 37°C, 5% CO 2 , and saturated humidity. After washing two times with icecold phosphate-buffered saline (PBS), 1 × 10 6 cells from each group were digested with trypsin and centrifuged for 5 min at 1000 × g, and the supernatant was discarded. For apoptosis, 500 μl of binding buffer was used to gently resuspend the cells. Then, 5 μl each of annexin V Fluorescein Isothiocyanate-FITC and propidium iodide (PI) was added to the cell suspension. After mixing, the cells were stained for 15 min at room temperature and analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). For cell cycle progression, the cells were washed with ice-cold PBS, fixed with 70% ethanol, centrifuged, and collected. The pelleted cells were washed with PBS and resuspended in 500 μl of PI staining solution. The cells were stained for 30 min at 4°C avoiding light and analyzed using a BD FACSCalibur flow cytometer. The data were analyzed with ModFit 4.0 (Verity Software House, Washington, USA).
6
Cell Cycle Analysis of Adipose-Derived MSCs
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Human adipose-derived MSCs were detached with 0.1% trypsin, 0.02% EDTA, washed twice in DPBS and centrifuged. The pellet was suspended in 0.01% nonidet P-40, 10 mg/mL RNase, 0.1% sodium citrate and 50 μg/mL propidium iodide (PI), for 30 min at room temperature in the dark. Propidium iodide fluorescence was analyzed using a Bryte HS flow cytometer (Bio-Rad, Hercules, CA, USA) equipped with Hg lamp and analyzed with ModFit 4(Verity Software House, Topsham, ME, USA) software.
7
DNA Content Analysis of Cell Lines
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For DNA content analysis on cell lines, the Vindeløv method was used (Vindelov et al. 1983) . In short, cells were harvested by trypsin/EDTA (Corver et al. 1995) (link), counted and 2 × 10 6 cells were divided over two polystyrene tubes. Cells were pelleted and to one tube, trout red blood cells (TRBC) were added as internal reference for DNA content. Next, solutions A, B and C, containing propidium iodide (PI), were added sequentially as described by the method. Samples were stored at 4°C and analyzed next day.
For DNA content of clinical samples, our in-housedeveloped multiparameter method was used (Corver et al. 2005 ). An LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) was used for collecting FITC, APC and/or PI fluorescence using a 530/30-, 670/14-and 610/20-nm band pass filter, respectively. 488-nm and 635-nm laser lines were used for excitation. Data were analyzed using WinList 8 and ModFit 4 (Verity Software House, Topsham, ME, USA).
For DNA content of clinical samples, our in-housedeveloped multiparameter method was used (Corver et al. 2005 ). An LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) was used for collecting FITC, APC and/or PI fluorescence using a 530/30-, 670/14-and 610/20-nm band pass filter, respectively. 488-nm and 635-nm laser lines were used for excitation. Data were analyzed using WinList 8 and ModFit 4 (Verity Software House, Topsham, ME, USA).
8
Cell Cycle Analysis of hPSCs
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hPSCs were carefully dissociated into single-cell suspensions using Accutase solution, washed twice with PBS, and then fixed overnight with cold 70% ethanol. The fixed cells were washed twice with PBS, followed by RNase (Sigma, 100 µg/mL) treatment and propidium iodide (Sigma, 50 µg/mL) staining for 30 min at 37 °C. Approximately 1×106 cells were analyzed using a FACSCanto II (Becton Dickinson) to determine the cell cycle distribution pattern. The percentages of cells in G1, S and G2/M phases of the cell cycle were analyzed using ModFit 4.0 software (Verity Software House).
9
Cell Cycle Analysis of Irradiated BAMC
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BAMC were isolated from femurs of 6-week-old WT and p53-null mice (n = 10) as described66 (link). Growing cells were irradiated with 10 Gy (second passage). In all, 24 h later cells were fixed in 70% ethanol at 4°C overnight. Cells were incubated in PBS with 50 μg/ml PI (BioLegend Inc., San Diego, CA, USA) and 100 μg RNase A (Invitrogen, Carlsbad, CA, USA). Data were acquired on an LSRII Fortessa FACScan flow cytometer (Becton Dickinson), stored in Listmode format, and analyzed using ModFit 4.0 software (Verity Software House). Scatter of the cells and doublet discrimination were criteria used prior to analyzing the DNA content of PI-stained cells.
10
Cell Apoptosis and Cell Cycle Analysis
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After transfection for 24 h, cell apoptosis was evaluated by staining with Annexin V and PI (Dojindo Molecular Technologies Inc., Shanghai, China) for 15 min at room temperature in the dark, followed by flow cytometry within 1 h (BD Biosciences, Erembodegem, Belgium). Cell apoptosis was analysed by Flowjo 7.6 software (TreeStar Inc., Ashland, OR, USA). After transfection for 48 h, cells were collected and fixed in 70% cold ethanol for at least 12 h at 4 °C. Cells were stained with 50 μg/ml propidium iodide (BD Biosciences, Erembodegem, Belgium) at room temperature for 30 min in the dark, and the cell cycle was analysed using the FACS Calibur system (BD Biosciences, Erembodegem, Belgium) and ModFit 4.0 software (Verity Software House, Topsham, ME, USA).
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