Each group of cells was seeded at 3×105 cells/well in six-well plates and cultured until 85% confluence was reached. The cells were washed three times with PBS, prior to collection by centrifugation (Allegra X-22R, Beckman Coulter, Miami, FL, USA) at 1,000 × g for 5 min. The cell pellets were subsequently resuspended in 1 ml PBS, fixed in 70% ice-cold ethanol and kept in a freezer for >48 h. Prior to flow cytometric analysis, the fixed cells were centrifuged at 1,000 × g for 5 min, washed twice with PBS, and resuspended in PI staining solution (Sigma-Aldrich), containing 50 µl/ml PI and 250 µg/ml RNase A (Sigma-Aldrich). The cell suspension, which was protected from the light, was incubated for 30 min at 4°C and analysed by fluorescence-activated cell sorting (FACS) using a BD FACSAria cell sorter (BD Biosciences, Frankin Lakes, NJ, USA). A total of 20,000 events were acquired for analysis using CellQuest software version 2.0 (BD Biosciences).
Cell quest software version 2
Manufactured by BD
Sourced in United States
Cell Quest software version 2.0 is a laboratory software designed for flow cytometry data analysis. It provides core functionality for basic processing and visualization of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facsvantage se, by BD (2 mentions)
Facsaria cell sorter, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Allegra x 22r, by Beckman Coulter (1 mentions)
Pi staining solution, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Polybrene, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Annexin 5 fitc pi cell apoptosis detection kit, by Meilun (1 mentions)
Cytoflex3l8c, by Beckman Coulter (1 mentions)
7 protocols using cell quest software version 2
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Viability and Apoptosis Analysis
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The determination of cell viability was done as described earlier [40 (link)]. Further, the flow cytometry was used to study the cell cycles (BD Corporation, Franklin Lakes, NJ, and USA) and the measurements of the apoptotic percentage were considered from sub-G0/G1 fraction. The data analysis was done by Cell Quest software version 2.0 (BD).
3
Downregulating miR-21 in SKM-1 Cells
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To down-regulate miR-21 in SKM-1 cells, the inhibitor of hsa-miR-21 lentivirus gene transfer vector encoding green fluorescent protein (GFP) was constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequence of the inhibitor of hsa-miR-21 5′-TAGCTTATCAGACTGATGTTGA-3′ was confirmed by sequencing (data not shown). The recombinant lentivirus of miR-21 inhibitor (LV-miR-21 inhibitor) and the control lentivirus (LV-NC, 5′-TTCTCCGAACGTGTCACGT-3′) were prepared and tittered to 1×108 transfection unit (TU)/ml. A total of ~0.5×105 SKM-1 cells were plated in each well in 24-well plates overnight at 37°C.
Following 24 h of culture, lentiviruses were diluted in 0.4 ml Iscove's Modified Dulbecco Medium (IMDM; Gibco; Thermo Fisher Scientific, Inc.) containing polybrene (5 µg/ml; Sigma-Aldrich; Merck KGaA) and added to the cells and incubated at 37°C for an additional 24 h, followed by incubation in 0.5 ml of fresh IMDM for another 24 h at 37°C, which was replaced with fresh IMDM and the cells were cultured for 48 h at 37°C. The lentivirus transduction efficiency of SKM-1 cells was determined by the detection of GFP signals by fluorescence microscopy (magnification, ×100) and flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) CellQuest software version 2.0 (BD Biosciences) 96 h following transduction. Data were analysed using CellQuest software (BD Biosciences).
Following 24 h of culture, lentiviruses were diluted in 0.4 ml Iscove's Modified Dulbecco Medium (IMDM; Gibco; Thermo Fisher Scientific, Inc.) containing polybrene (5 µg/ml; Sigma-Aldrich; Merck KGaA) and added to the cells and incubated at 37°C for an additional 24 h, followed by incubation in 0.5 ml of fresh IMDM for another 24 h at 37°C, which was replaced with fresh IMDM and the cells were cultured for 48 h at 37°C. The lentivirus transduction efficiency of SKM-1 cells was determined by the detection of GFP signals by fluorescence microscopy (magnification, ×100) and flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) CellQuest software version 2.0 (BD Biosciences) 96 h following transduction. Data were analysed using CellQuest software (BD Biosciences).
4
Cell Cycle Analysis with BI-69A11
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The cells were grown and treated with BI-69A11 for varying time points. Cells were fixed with 70% ethanol, treated with RNaseA (100 μg ml−1) and propidium iodide at a final concentration of 40 μg ml−1, and incubated for 45 min at 37 °C. Finally, the cells were analysed with the FACS Vantage SE (BD Corporation, Franklin Lakes, NJ, USA). Cell cycle was also analysed by using Annexin-FITC apoptosis detection kit (Sigma-Aldrich) as per manufacturer's protocol. Cell Quest software version 2.0 (BD) was used for data analysis.
5
Cell Cycle Analysis by Flow Cytometry
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Cells grown to 70-80% confluence were treated with GW627368X (9, 10, 10 μM for HeLa, SiHa and ME 180 respectively) for varying time points, 70% ethanol fixed, RNaseA (100 μg/ml) and propidium iodide at a final concentration of 40 μg/ml added and incubated for 45 min at 37 °C. Cells were analyzed with FACS Vantage SE (BD Corporation, Franklin Lakes, NJ, USA). Results obtained were analyzed with Cell Quest software version 2.0 (BD).
6
Cell Cycle Progression Analysis by Flow Cytometry
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A7r5 cells were seeded in a 6-well tissue culture plate at a density 2.5×105 cells/well. These cells were then incubated at 37°C with three different concentrations of OA (0, 50 or 800 µmol/l) for 24 h; 0 µmol/l OA (containing 1 mmol/l DMSO) served as a control. After treatment, cells were harvested using trypsin. The cells were centrifuged at 300 × g for 5 min at 25°C, washed twice with cold PBS and fixed with 75% cool ethanol at −20°C overnight (not <16 h). The fixed cells were then centrifuged at 200 × g for 5 min at 25°C and washed twice with cold PBS. Then, cells were stained with 50 µg/ml PI containing 10 µg/ml RNase A for 30 min on ice. Finally, the distributions of cells in different cell cycle phases were assessed using specific amounts of cellular DNA (2×105 cells/tube) and flow cytometry (CytoFLEX; Beckman Coulter, Inc.). In total, >10,000 cells were counted per sample, and DNA histograms for cell cycle analysis were analyzed using Cell Quest software version 2.0 (BD Biosciences). The percentage of cells in the G0/G1 phase, S phase and G2/M phase were analyzed, and all experiments were repeated in triplicate.
7
Annexin V-FITC/PI Apoptosis Assay
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Following incubation for 48 h, a AnnexinV-FITC/PI cell apoptosis detection kit (Meilunbio) was used to measure apoptotic cells. The cells (2x105) were centrifuged at 800 x g for 5 min at 4˚C, collected and washed with PBS twice at 4˚C. The cells (1x106/ml) were resuspended with 250 µl 1X binding buffer. The cells (100 µl) were subsequently added to a pipe with 5 µl Annexin V-FITC and 10 µl propidium iodide (PI; 20 µg/ml). The mixture was cultured at 25˚C for 15 min in darkness. The apoptotic rate was analyzed using a flow cytometer (Cytoflex3L8C; Beckman Coulter, Inc.) and the results were analyzed using Cell Quest software version 2.0 (BD Biosciences).
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