Cells were pulse labeled with 5-bromo-2′-deoxyuridine (BrdU) labeling reagent (Invitrogen, 1:100 in pre-warmed DMEM) for 1.5 hours and then fixed in 1% paraformaldehyde overnight at 4°C. Cells were permeabilized with 0.1% tritonX and subsequently treated with 0.3 mg/ml DNase (Sigma-Aldrich) in PBS for 30 minutes at 37°C. The cells were then incubated with anti-BrdU antibody (Invitrogen, 1:50 in PBS + 2% FBS), washed in PBS, and resuspended in PBS containing 2% FBS and 5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Fluorescence intensity of BrdU and DAPI positive cells expressing GFP expression was evaluated on a LSR II flow cytometer.
5 bromo 2 deoxyuridine brdu labeling reagent
Manufactured by Thermo Fisher Scientific
5-bromo-2'-deoxyuridine (BrdU) is a labeling reagent that can be incorporated into newly synthesized DNA during cell division. It is used to identify and quantify proliferating cells.
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Lab products found in correlation
Anti brdu antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
Flow cytometry system, by BD (1 mentions)
Pi solution, by Merck Group (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 mouse anti brdu, by Thermo Fisher Scientific (1 mentions)
2 protocols using 5 bromo 2 deoxyuridine brdu labeling reagent
1
Cell Proliferation Measurement by BrdU
2
ECFC Proliferation Assay with MPA
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ECFC (3-5x105) were exposed to either vehicle control or MPA 1 μM. After 3 days, cells were treated with 5’-bromo-2’-deoxyuridine (BrdU) labeling reagent (Invitrogen) for 1 hour at 37 °C with 5% CO2. Cells were stained using Alexa Flour 488 mouse anti-BrdU (Invitrogen) for 90 minutes at room temperature and 7-AAD (Life Technologies) for 15 minutes at room temperature. Samples were analyzed by flow cytometry on the LSRII 407nm laser and a minimum of 10,000 events was recorded per sample. Analysis was performed using FlowJo Single Cell Analysis Software vX.0.6.
ECFC (5-50x103) cells were seeded in 10 cm2 culture dish and incubated in EGM-2 medium with and without MPA for either 3 or 7 days. Cells were collected, washed, fixed with 3.7% formaldehyde for 10 minutes at room temperature, washed again, and re-suspended in 25 μg/ml PI solution (Sigma Aldrich) for 15 minutes in the dark. The percentage of cells at the G0/G1, S and G2/M phases of the cell cycle were analyzed on a flow cytometry system (BD Biosciences, San Jose, CA) and FlowJo software (TreeStar).
ECFC (5-50x103) cells were seeded in 10 cm2 culture dish and incubated in EGM-2 medium with and without MPA for either 3 or 7 days. Cells were collected, washed, fixed with 3.7% formaldehyde for 10 minutes at room temperature, washed again, and re-suspended in 25 μg/ml PI solution (Sigma Aldrich) for 15 minutes in the dark. The percentage of cells at the G0/G1, S and G2/M phases of the cell cycle were analyzed on a flow cytometry system (BD Biosciences, San Jose, CA) and FlowJo software (TreeStar).
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