Prior to the determination of cell cycle distribution, the infected or/and transfected cells were grown until they achieved 30–40% confluence. The cells were then incubated in L15 medium at 28 ℃ for 6 h in the presence/absence of anti-p53 blocking antibody (30 ng/ml). The cells were then treated with 2 mM thymidine (Sigma Aldrich, St. Louis, MO, USA) for 12 h, after which they were transferred to growth medium for 10 h. This was followed by a secondary thymidine block of 12 h, after which the cells were transferred to growth medium. The cells were harvested at pre-determined times and then fixed with 70% cold ethanol and stained with 50 mg/ml propidiumiodide (Sigma, St Louis, Mo, USA), which was followed by RNase A (Sigma) treatment for 30 min at room temperature. DNA content was analyzed by a FACscan cell analyzer (BD Biosciences, San Jose, CA, USA) equipped with Cell quest software (BD Biosciences, San Jose, CA, USA). The population of cells in each phase was determined using ModFit LT software (BD Biosciences, San Jose, CA, USA).
Facscan cell analyzer
Manufactured by BD
Sourced in United States
The FACScan cell analyzer is a flow cytometry instrument used for analyzing and sorting cells. It is designed to measure and analyze multiple physical characteristics of cells or particles as they flow in a fluid stream through a beam of light.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (3 mentions)
Rnase a, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Modfit lt software, by BD (1 mentions)
Thymidine, by Merck Group (1 mentions)
12 well tissue culture plate, by BD (1 mentions)
Pi rnase staining buffer, by Merck Group (1 mentions)
Dulbecco s pbs, by Lonza (1 mentions)
Propidium iodide, by Carl Roth (1 mentions)
Fcs express 4 flow cytometry software, by De Novo Software (1 mentions)
5 protocols using facscan cell analyzer
1
Cell Cycle Analysis of Transfected/Infected Cells
2
Adenovirus Transduction of Immature Dendritic Cells
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Immature d4 DCs were seeded in 12-well tissue culture plates (BD Falcon) at a concentration of 1 × 106 cells/well in 250 μL medium supplemented with either 800 U/mL GM-CSF and 500 U/mL IL-4 (for iDCs) or the maturation cocktail consisting of 400 U/mL IL-1β, 2000 U/mL IL-6, 20 ng/mL TNF-α, and 2 μg/mL PGE2, in addition to IL-4 and GM-CSF (for mDCs). When indicated, 0.1 ng/mL LPS were used instead of the cytokine cocktail, which was added to the cell culture after the transduction. Adenovirus was added to the cells at 500 TCID50/cell in a final volume of 250 μL medium without cytokines. For cotransduction of two adenoviruses, the final volume was halved to 125 μL medium without cytokines, also resulting in a final infection volume of 500 μL. After 1.5 hours of incubation at room temperature, 2 mL of growth medium replenished with cytokines as described before was added per well. To determine transduction efficacy, cells were transduced with Ad5TL and the percentage of living green fluorescent cells was assessed by flow cytometric analysis with a FACScan cell analyzer (BD Biosciences, NJ, US). Only experiments that yielded transduction efficiencies of more than 70% were evaluated and are shown.
3
Cell Cycle Analysis by Flow Cytometry
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Infected cells were cultured for several days at a confluence of less than 80%. Cells were harvested and suspended in 1X PBS and fixed with cold ethanol at a final concentration of 70%. The cells were then stained with 50 mg/mL propidium iodide (Sigma‐Aldrich) followed by RNase A (Sigma‐Aldrich) treatment for 30 min at room temperature. DNA content was analyzed using a FACScan cell analyzer (BD Biosciences, San Jose, CA, USA) equipped with CellQuest software (BD Biosciences). Finally, the cell population in each cell cycle phase was determined.
4
Cell Cycle Analysis by Flow Cytometry
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After the cancer cells were treated with various drug combinations, they were fixed and re-suspended in a propidium iodide (PI)/RNase staining buffer (Sigma). The stained cells were then analyzed for DNA content using a FACScan cell analyzer equipped with Cellquest software (BD Biosciences).
5
Flow Cytometry of Dendritic Cells
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Cells were stained with specific mAb or appropriate isotype controls for 30 min at 4°C in FACS buffer (Dulbecco's PBS [Lonza] containing 2% FCS [PAA/GE Healthcare]), washed twice, and finally resuspended in cold FACS buffer containing 0.1 μg/mL propidium iodide (PI) (Carl Roth, Karlsruhe, Germany). Stained cells were immediately analyzed with a FACScan cell analyzer (BD Biosciences). Cell debris and dead cells were excluded from the analyses by gating on proper forward and sideward light scatter and on PI negative cells. Also, percentages of PI positive cells were calculated using this gating strategy. A minimum of 104 living cells was measured for each sample and results were analyzed using FCS Express 4 Flow Cytometry Software (De Novo Software, CA, US). The following monoclonal antibodies (all from BD Biosciences) were used to determine the phenotype of DCs: PE-labeled mouse anti-human CD25 (M-A251), CD80 (L307.4), CD83 (HB15e), CD86 (IT2.2), HLA-ABC (G46-2.6), and HLA-DR (G46.6). Isotype mAb controls (all from BD Biosciences) used but not shown were IgG1-PE (MOPC-21), IgG2a-PE (G155-178), and IgG2b-PE (27-35).
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