Transfected OCI-Ly7 cells (1×103 cells/well) were seeded into a 96-well plate and cultured for 48 h at 37°C. For cell cycle analysis, transfected OCI-Ly7 cells were collected and fixed with 70% ethanol at 4°C overnight. After washing with PBS, the transfected OCI-Ly7 cells were incubated with 1 mg/ml RNase A for 20 min at 37°C, followed by staining with propidium iodide (PI) and 1% Triton X-100 for 20 min at 4°C. The experimental data were collected using a flow cytometer (BD FACScan; Becton, Dickinson and Company). For apoptosis analysis, cells were washed with PBS and resuspended with 100 µl binding buffer, followed by addition of 10 µl PI/FITC-Annexin V (Roche Diagnostics) and incubation at room temperature for 1 h in the dark. Flow cytometry (BD FACScan; Becton, Dickinson and Company) was used to detect apoptosis within 1 h. The cell cycle distribution and apoptosis of transfected OCI-Ly7 cells were analyzed by Cell Quest acquisition and analysis software (V6.0; BD Biosciences).
Facscan
Manufactured by BD
Sourced in United States, Germany, China, United Kingdom, Italy, Japan, Spain, France, Canada, Switzerland, Jersey, Macao
The FACScan is a flow cytometry instrument manufactured by BD. It is designed to analyze and sort cells or particles in a fluid stream. The FACScan uses laser technology to detect and measure the physical and fluorescent characteristics of individual cells or particles as they pass through the instrument's flow cell.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (344 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (105 mentions)
Propidium iodide, by Merck Group (99 mentions)
Cycletest plus dna reagent kit, by BD (74 mentions)
Cellquest, by BD (73 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by BioLegend (48 mentions)
Facscalibur, by BD (43 mentions)
Propidium iodide, by BD (41 mentions)
Rnase a, by Merck Group (40 mentions)
Annexin 5 fitc, by BD (38 mentions)
Cellquest pro software, by BD (32 mentions)
Cellquest pro, by BD (31 mentions)
Propidium iodide, by Thermo Fisher Scientific (27 mentions)
Cellquest 3, by BD (20 mentions)
Modfit software, by BD (19 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (14 mentions)
Sytox green, by Thermo Fisher Scientific (13 mentions)
Propidium iodide, by Beyotime (13 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (12 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (12 mentions)
3 082 protocols using facscan
1
Cell Cycle and Apoptosis Analysis of Transfected OCI-Ly7 Cells
2
Cell Cycle and Apoptosis Analysis in ESCC
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For cell cycle assays, ESCC cells were harvested 24 h after serum starvation and fixed in 80% ice-cold ethanol in phosphate-buffered saline (PBS) after washing in ice-cold PBS. Then cells were incubated at 37 °C for 30 min; bovine pancreatic RNAase (Sigma) was added at a final concentration of 2 mg/ml and 20 mg/ml of PI (Sigma-Aldrich, USA) for 30 min at room temperature. Cell cycle distribution was flow cytometrically determined using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). For apoptosis assays, a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (Dojindo, Kumamoto, Japan) was used according to the manufacturer’s instructions. Briefly, cells were collected by mild trypsinization, washed twice in cold PBS, stained with FITC-Annexin V and PI on ice for 5 min, and subjected to flow cytometry using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Cell Cycle and Differentiation Analysis
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Cell-cycle profile was analyzed using flow cytometry. Briefly, 2 × 106 cells were washed with PBS, fixed in 80% ethanol and resuspended in 50μg/ml propidium iodide (Sigma) and 125U/ml RNase A (Sigma). For cell cycle profile, DNA content was analyzed by flow cytometry using FACScan and Cell Quest Software (Becton Dickinson, USA). For detection of cell differentiation antigen cd11b and cd114, 1 × 106 cells were washed twice with PBS, incubated with PE-conjugated cd11b or cd114 antibody or PE-conjugated IgG isotype control antibody at 4°C for 30 min and analysed by flow cytometry using FACScan and Cell Quest Software (Becton Dickinson, USA) [12 (link), 13 (link), 29 (link)].
4
Comparative Analysis of AFP Promoter Variants
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The fluorescence-activated cell sorting (FACS) analysis was conducted to assess the specificity and transcriptional activity of modified AFP promoters with HREs located upstream. Cells were transduced with dAd/CMV-GFP, dAd/AFPm-GFP, dAd/a2bm-GFP, dAd/a2bSm-GFP, dAd/Ha2bm-GFP, or dAd/Ha2bSm-GFP (Huh7 and A549 at 40 MOI, HepG2 at 30 MOI, Hep3B and Hep1 at 20 MOI, and BJ at 100 MOI). At 48 h post transduction, cells were harvested using a dissociation solution (Sigma, St. Louis, MO) and washed with phosphate buffered saline (PBS). GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson, East Rutherford, NJ). Data were collected from 10,000 cells and analyzed with the CellQuest software (BD Biosciences Immunocytometry Systems, San Jose, CA).
To compare the promoter strength of full length hAFP promoter and a2bm promoter, Hep3B and Huh7 cells were transduced with replication-incompetent Ad expressing GFP under the control of hAFP promoter (dAd/hAFP-GFP) or dAd/a2bm-GFP at 40 or 50 MOI, respectively. GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson) as described above.
To compare the promoter strength of full length hAFP promoter and a2bm promoter, Hep3B and Huh7 cells were transduced with replication-incompetent Ad expressing GFP under the control of hAFP promoter (dAd/hAFP-GFP) or dAd/a2bm-GFP at 40 or 50 MOI, respectively. GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson) as described above.
5
Analyzing Yeast Cell Viability and Mitochondrial Potential
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Propidium iodide (PI) staining of S. cerevisiae cells was performed by incubating 1 mL of cell culture with 0.0005% PI (Sigma-Aldrich) at RT in the dark for 2 min. Stained cells were observed by fluorescence microscopy. A 1:10 dilution in PBS of the previous cell suspension was analyzed by flow cytometry using a FACScan instrument (Becton Dickinson) with excitation at 488 nm and the FL3 detector using the 650/LP long-pass filter.
Mitochondrial membrane potential was evaluated by flow cytometry in cells stained with rhodamine 123 (Rh 123). One milliliter of cell suspension was treated with Rh 123 (Sigma-Aldrich) at a final concentration of 5 μg/mL and aerobically incubated at 30°C in the dark for 30 min. Then, cells were stained with PI as stated above. Samples were diluted 1:10 in PBS and analyzed by flow cytometry using a FACScan instrument (Becton Dickinson) with excitation at 488 nm, the FL1 detector with a 530/30 band pass filter for Rh 123 analysis, and the FL3 detector with the 650/LP long-pass filter for PI analysis. PI-positive cells were excluded from the analysis of Rh 123-derived fluorescence.
Mitochondrial membrane potential was evaluated by flow cytometry in cells stained with rhodamine 123 (Rh 123). One milliliter of cell suspension was treated with Rh 123 (Sigma-Aldrich) at a final concentration of 5 μg/mL and aerobically incubated at 30°C in the dark for 30 min. Then, cells were stained with PI as stated above. Samples were diluted 1:10 in PBS and analyzed by flow cytometry using a FACScan instrument (Becton Dickinson) with excitation at 488 nm, the FL1 detector with a 530/30 band pass filter for Rh 123 analysis, and the FL3 detector with the 650/LP long-pass filter for PI analysis. PI-positive cells were excluded from the analysis of Rh 123-derived fluorescence.
6
Cell Cycle Analysis and P-gp Activity Assay
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Cells were seeded into 6-wells for overnight. After adherence, cells were treated with indicated concentration of MPT0G066 for different time intervals and then collected by trypsinization, fixed with 75% (v/v) ethanol at −20 °C overnight. After centrifugation, cells were incubated in phosphate-citric acid buffer (NaHPO4 0.2 M, citric acid 0.1 M (pH 7.8)) for 20 minutes at room temperature. Then, cells were centrifuged and resuspended with 0.5 mL PI solution (Triton X-100 0.1%, RNase 100 μg/mL and PI 80 μg/mL). DNA content was analyzed with the FACScan and CellQuest software (Becton Dickinson).
For p-glycoprotein (p-gp) activity assay, multi-drug resistant NCI/ADR cells were co-treated with indicated agents and 10 μM rhodamine 123 for 60 min. Then, cells were washed by PBS and collected by trypsinization. The results were detected with the FACScan and CellQuest software (Becton Dickinson).
For p-glycoprotein (p-gp) activity assay, multi-drug resistant NCI/ADR cells were co-treated with indicated agents and 10 μM rhodamine 123 for 60 min. Then, cells were washed by PBS and collected by trypsinization. The results were detected with the FACScan and CellQuest software (Becton Dickinson).
7
Cell Cycle Analysis by Flow Cytometry
8
Apoptosis and Cell Cycle Analysis
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Transfected Hep 3B and Huh7 cells were processed with an Apoptosis Detection Kit (Beyotime, China), according to the manufacturer’s instructions. Briefly, transfected cells were washed twice with cold PBS. After incubation with 5 μL of FITC-Annexin V and 5 μL propidium iodide for 20 min in the dark, apoptosis of Hep 3B and Huh7 was detected using a flow cytometer (FACScan; BD Biosciences, USA).
For cell cycle analyses, cells were collected and incubated with 70% ethanol at 4°C overnight for fixation. The cells were washed twice with PBS and incubated with 100 μg/mL RNase A and 50 μg/mL propidium iodide for 1 h at 37°C. The percentage of cells in each phase of the cell cycle was then measured by flow cytometry (FACScan; BD Biosciences, USA).
For cell cycle analyses, cells were collected and incubated with 70% ethanol at 4°C overnight for fixation. The cells were washed twice with PBS and incubated with 100 μg/mL RNase A and 50 μg/mL propidium iodide for 1 h at 37°C. The percentage of cells in each phase of the cell cycle was then measured by flow cytometry (FACScan; BD Biosciences, USA).
9
CD46 Expression and Apoptosis Analysis
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CD46 expression and the apoptosis of cells were determined by flow cytometry. To measure CD46 expression, the cells were harvested and incubated with a fluorescein isothiocyanate–labeled monoclonal mouse antihuman CD46 or control antibodies (BD Biosciences, Pharmingen) for 0.5 hour on ice. Then cells were washed twice with cold PBS and 1 × 104 cells per sample were analyzed using a FACScan (BD Biosciences, San Jose, CA). For sub-G1 analysis, SiHa cells were seeded in 6-well plates and infected with MV-Edm at an MOI of 1. All the cells were harvested at 24, 48, and 72 hours after infection and fixed in ice-cold 70% ethanol for at least 1 hour. Then the cells were rinsed twice with PBS and incubated for 30 minutes at room temperature in 1 ml PBS containing 50 μg propidium iodide (Sigma-Aldrich, St Louis, MO), 0.1% Triton X-100, 1 mmol/l EDTA, and 0.5 mg RNaseA. The stained cells were subjected to flow cytometric analysis with a FACScan (BD Biosciences, San Jose, CA). Fragmented, apoptotic nuclei were recognized by their sub-G1 DNA content. The proportion of cells in sub-G1 phase was determined for each group.
10
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells were harvested and fixed overnight in 70% (v/v) ethanol at 4 °C. Subsequently, cells were stained with 1 mg/mL propidium iodide at 37 °C for 30 min after mixing with 10 mg/mL RNase and assessed immediately by flow cytometry (FACScan; BD Biosciences, Shanghai, China). To analyse cell apoptosis, cells were harvested and washed twice with ice-cold PBS, followed by resuspension in the binding buffer. Subsequently, 5 μL of Annexin V-APC and 10 μL of 7-AAD were added to 100 μL of the cell suspension at 4 °C for 15 min in the dark. Cell apoptosis was determined by flow cytometry (FACScan; BD Biosciences, Shanghai, China). Data on the cell cycle and apoptosis were analysed by FlowJo 7.6 (Treestar, Woodburn, OR, USA).
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