Cell cycle distribution was analyzed using flow cytometry. Briefly, 1 × 106 cells were harvested, washed in PBS and then fixed in 70% alcohol for 30 min at 4°C. After 3 washes in cold PBS, the cells were resuspended in 1 mL PBS solution containing 50 μL of 1 mg/mL PI and 1 unit DNase‐free RNase for 30 min at 37°C. The samples were then analyzed to determine the DNA content using a FACScan analyzer (Beckman Coulter Inc., Fullerton, CA, USA). For cell death analysis, Annexin V‐FITC/PI staining was carried out by incubating the cells for 15 min at room temperature (RT) in a binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5 mmol/L CaCl2, pH 7.4) containing saturated concentrations of Annexin V‐FITC and PI. After incubation, the cells were pelleted and analyzed in a FACScan analyzer (Beckman Coulter Inc.).
Facscan analyzer
Manufactured by Beckman Coulter
Sourced in United States
The FACScan analyzer is a flow cytometry instrument designed for the analysis of cells and particles. It is capable of detecting and measuring various physical and fluorescent properties of individual cells or particles as they pass through a laser beam. The FACScan provides data on parameters such as size, granularity, and the presence of specific fluorescently-labeled molecules within the sample.
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Lab products found in correlation
3 protocols using facscan analyzer
1
Cell Cycle and Apoptosis Analysis
2
Immunofluorescence Analysis of Rat GEC Cells
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Rat GEC monolayers incubated with Kgp or TCLK-treated Kgp were gently dispersed and resuspended at a final concentration of 3×106 cells/ml. After washing with PBS, cells were fixed with OptilyseC (Becton Dickinson, Franklin Lakes, NJ) containing 0.2% Triton X-100 (Sigma-Aldrich). Next, cells were washed with 0.2% Triton X-100 in PBS (washing buffer) and incubated with anti-cytokeratin-6 antibody (20 μg/ml) or with the same concentration of non-immune serum for 1 h in washing buffer, followed by incubation with FITC-conjugated secondary antibody (ICN Pharmaceuticals, Aurora, OH) for 30 min. Fluorescence was analyzed with a FAC-Scan analyzer (Beckman Coulter, Fullerton, CA).
3
RAGE Expression Analysis in OLC-1 Cells
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OLC-1 monolayers were gently dispersed and resuspended at a final concentration of 3 × 106 cells/mL and FACS was performed as described previously [38 ] with slight modifications. After washing with PBS, cells were fixed with OptilyseC (Becton Dickinson, Franklin Lakes, NJ, USA). Next, cells were washed with PBS and incubated with the RAGE Ab or isotype-matched control (2 μg/mL), at 4°C for 1 h, followed by the fluorescein isothiocyanate (FITC-) conjugated secondary Ab (ICN Pharmaceuticals, Aurora, OH, USA) for 30 min in the dark. Fluorescence was analyzed with a FACScan analyzer (Beckman Coulter, Fullerton, CA, USA).
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