Cells were harvested after incubation with 30 μM bromodeoxyuridine (BrdU; Sigma–Aldrich) for 30 min, fixed in 1 ml of cold 70% ethanol and stored at 4°C. DNA was denatured with 2N HCl and 0.5% Triton X-100, then neutralized with 1 ml of 0.1M sodium borate pH8.5. The cell pellet was resuspended in PBS, 0.5% Tween 20, 1% bovine serum albumin (BSA) with a mouse anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at room temperature for 1 h, followed by 1-h room temperature incubation with anti-mouse-488 (Alexa anti-mouse 488, Molecular Probes/Invitrogen). Samples were finally incubated with 10 μg/ml Propidium Iodide and analyzed on a S3 flow cytometer (Biorad). Data were analyzed using the program ProSort (Biorad) or FCS Express (DeNovo). When Propidium Iodide only staining was performed, cells were fixed in 1 ml of cold ethanol and resuspended in PBS, 0.1% Tween 20, 50 μg/ml Propidium Iodide, 5 μg/ml RNase A. After incubation at 37°C for 15 min, cells were analyzed by flow cytometry.
Prosort
Manufactured by Bio-Rad
The ProSort is a high-performance cell sorter designed for precise and efficient cell separation. It utilizes advanced flow cytometry technology to rapidly sort and collect cells based on user-defined parameters. The ProSort provides reliable and reproducible results, making it a versatile tool for a wide range of applications in cell biology and life science research.
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2 protocols using prosort
1
BrdU Incorporation and Cell Cycle Analysis
2
BrdU Pulse-Chase Assay for DNA Replication
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The U2OS and BJ-hTERT cells were pulsed with 30 µM BrdU ((Sigma Aldrich) for 30 min or 1 h and the DNA replication was followed after its withdrawal, respectively, for 0–6–9 or 12 h in cells transfected with scrambled siRNA or after silencing of POLL. The cells were then fixed in 1 mL of cold 70% ethanol and stored at 4 °C overnight. DNA was denatured with 2N HCl/0.5% Triton X-100 and neutralized with 1 mL of 0.1M sodium borate pH8.5. After centrifugation at 500g for 10 min, the cell pellet was resuspended with a mouse anti-BrdU antibody (1:100; Becton Dickinson,), diluted in 1% BSA, PBS 0.5% Tween 20, and incubated for 1 h at room temperature. Cells were then centrifuged at 500 g for 10 min and the pellet was resuspended with anti-mouse-488 (1:500; Alexa anti mouse 488, Molecular Probes/Invitrogen) and incubated for 1 h at room temperature. Samples were finally incubated with 10 ug/mL Propidium Iodide and analysed on a S3 flow cytometer (Bio-rad). Data were analysed using the program ProSort (Bio-rad) or FCS Express (DeNovo).
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