Cells were fixed in cold 70% ethanol at the indicated times after 30 μM bromodeoxyuridine (BrdU; Sigma-Aldrich) incubation for 30 min and stored at 4°C. DNA was denatured with 2N HCl and 0.5% Triton X-100 and then neutralized with 0.1 M sodium borate (pH 8.5). The pellet was resuspended with 0.5% Tween 20 and 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), anti-BrdU (Becton Dickinson, Franklin Lakes, NJ, USA) was added for 1 h, followed by 1 h of anti-mouse-488 (Alexa anti-mouse 488, Molecular Probes/Invitrogen). The samples were incubated with 10 μg/ml propidium iodide and analysed on a S3 flow cytometer (Biorad, Hercules, CA, USA). The plots were analysed using FCS Express software (De Novo Software). For single propidium iodide staining, the cells were fixed as above before resuspension in PBS-0.1% Tween-20, 50 μg/ml propidium iodide, 5 μg/ml RNase A. After incubation for 15 min, the samples were analysed by FACS. Cell cycle distribution was fitted to a single cycle model with FCS Express.
Anti mouse 488 alexa anti mouse 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse-488 is a fluorescently-labeled secondary antibody that binds to mouse primary antibodies. It is conjugated to the Alexa Fluor 488 dye, which emits green fluorescence upon excitation. This product can be used for various immunodetection applications, such as flow cytometry, immunofluorescence, and Western blotting.
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2 protocols using anti mouse 488 alexa anti mouse 488
1
BrdU Incorporation and Cell Cycle Analysis
2
BrdU Incorporation and Cell Cycle Analysis
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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.
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