To detect the proportion of cells in different stages of the cell cycle and apoptotic cells, flow cytometry (FACSCalibur; BD Biosciences) was conducted. A total of 3 days post-transfection, the A549 and H1299 cells were collected, rinsed with PBS and treated with 75% ethanol at 4°C for 2 h. After washing with PBS, the cells were incubated with 200 µg/ml RNAse (cat. no. 10109134001; Sigma-Aldrich; Merck KGaA) for 20 min at room temperature and stained with 1 µg/ml propidium iodide (PI; BD Biosciences) in 1 ml PBS at 4°C for 30 min. Subsequently, the cell cycle was measured by flow cytometry. Cell apoptosis was detected after washing with 500 µl PBS. Briefly, 10% Annexin V-FITC (BD Biosciences) and 50 µg/ml PI staining was performed for 15 min in the dark at room temperature. To promote apoptosis, A549 cells were treated with paclitaxel (Shanghai YuanYe Bio-Technology Co. Ltd.) at a final concentration of 50 nM, followed by incubation for 18 h at 37°C. In addition, H1299 cells were treated with erlotinib (MAYA Reagent) at a final concentration of 2 µM and were incubated for 18 h at 37°C. Apoptosis was measured by flow cytometry using a FACSCalibur analysis system (BD Biosciences). Data were analyzed with Modfit 2.0 (Verity Software House, Inc.).
Modfit 2
Manufactured by Verity Software House
Sourced in United States
ModFit 2.0 is a software application designed for the analysis and modeling of flow cytometry data. The core function of the software is to provide users with tools for the identification and quantification of cell subpopulations within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Annexin 5 fitc, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Paclitaxel, by Yuanye Bio-Technology (1 mentions)
Facsdiva, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Facscan, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Muse flow cytometer, by Merck Group (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
8 protocols using modfit 2
1
Cell Cycle and Apoptosis Analysis by Flow Cytometry
2
Flow Cytometric Analysis of Samples
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Flow cytometric analysis was performed using a FACS Canto flow cytometer (Becton Dickinson, San Diego, CA, USA) equipped with 488 nm (blue) and 633 (red) lasers. Data acquisition and analysis were performed using FACSDiva (Becton Dickinson) and ModFit 2.0 (DNA Modelling System, Verity Software House, Inc., Topsham, ME, USA). Samples were run in triplicate and 10,000 events were collected for each replica. Data were the average of three experiments, with errors under 5%.
3
Cell Cycle Analysis by PI and BrdU
4
Cell Cycle Analysis by Flow Cytometry
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Flow cytometry was used to observe the cell cycle distribution. Briefly, HL-60 cells (4x105) were collected after 48 h of transfection and were stained with 50 µg/ml PI (Dojindo Molecular Technologies, Inc.) for 15 min at room temperature. Cell cycle distribution and sub-G1 DNA content were analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc.).
5
Apoptosis Evaluation by Flow Cytometry
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Cell apoptosis was evaluated through flow cytometry using a cellular Annexin V-FITC/PI Kit [cat. no. 70-AP101-100; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.] according to the manufacturer's protocol. Cells were plated into six-well plates at a density of 4x105 cells/well. After 48 h of transfection, cells (4x105 cells/well) were incubated with 200 µl binding buffer and double stained with Annexin V-FITC and PI at 4˚C in the dark for 20 min. The cells were then assessed using BD FACSCalibur™ (BD Biosciences) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc.).
6
PM2.5-Induced Cell Cycle Analysis
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Cells were treated with PM2.5 suspension for 48 h, centrifuged at 13,000 × g for 3 min at 4°C and washed twice with PBS. Cells were resuspended with PBS, and 2 volumes of 5 ml ethanol were slowly added and mixed, stationary set was at 4°C. The cells were washed twice with PBS, resuspended in 1 ml of PI solution and incubated at room temperature for 30 min. The cell cycle was detected by flow cytometry (Muse™ flow cytometer; Merck Millipore, Burlington, MA, USA) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc., Berlin, Germany).
7
Flow Cytometric Analysis of DNA Content
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Adenovirus-infected C3H10T1/2 cells were cultivated as described above, harvested, washed with PBS, and fixed with 70% ice-cold ethanol at 4 °C for 48 h. The fixed cells were washed with 100 µg/mL Ribonuclease H in PBS, incubated at 37 °C for 30 min, stained with 20 µg/mL propidium iodide in PBS in the dark for 30 min, and subsequently analyzed at 488 nm on a flow cytometer (BD LSR, Franklin Lakes, New Jersey, USA). DNA was quantitated by using Modfit 2.0 software (Verity Software House, Topsham, Maine, USA).
8
Cell Cycle Analysis in Prostate Cells
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For cell cycle evaluation, scramble shRNA or shSPOCK1-treated PC3 cells and vector or SPOCK1 plasmid-transfected RWPE-1 cells were trypsinized and fixed in ice-cold methanol for 20 minutes. After RNase treatment, the cells were treated with protease inhibitor (PMSF, 50 μg/mL) for 30 minutes, and the fluorescence intensities were detected using the FC500 flow cytometer (Beckman, Pasadena, CA, USA) with FL-1H filter. Furthermore, Modfit 2.0 software (Verity Software House Inc, Topsham, ME, USA) was used to quantify the cell numbers in G0/G1, S, and G2/M phases.
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