BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells. Print this protocol BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal.
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How is BrdU detected in the assay?
Using an anti-BrdU antibody, S phase cells can then be detected. The standard BrdU protocol uses acid treatment to unwind the DNA to help the anti-BrdU antibody access DNA incorporated BrdU. The harsh acid treatment doesn’t allow using this method in combination with immunophenotyping or with GFP labelled cells.
Why use DNase I pre-treatment?
Whole-mount BrdU staining of proliferating cells by DNase treatment: application to postnatal mammalian retina Biotechniques . 2006 Jan;40(1):29-30, 32. doi: 10.2144/000112094.
What is the DNA denaturing step after BrdU labeling?
Anti-BrdU Staining Using DNAse with Surface and Fluorescent Proteins . Note: We offer two protocols here depending on what your experiment requires. Ethanol treatment is usually harsher toward any other fluors or fluorescent proteins that may be present in your sample. As such, the DNAse method may be gentler under those conditions . Protocol Steps
Does excess DNase I treatment result in signal degradation?
During the BrdU assay, BrdU is incorporated into replicating DNA and can be detected using anti-BrdU antibodies. After BrdU labeling, an additional DNA hydrolysis step (sometimes referred to as a DNA denaturing step) may be required after fixation and permeabilization to allow the anti-BrdU antibody access to the BrdU within the DNA.
What is the purpose of BrdU?
Bromodeoxyuridine (BrdU) is a thymidine analog that incorporates DNA of dividing cells during the S-phase of the cell cycle. As such, BrdU is used for birth dating and monitoring cell proliferation.
What is BrdU staining?
BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells. BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal.
How do you use BrdU?
Label cells with BrdUCulture cells in appropriate vessel for microscopy.Remove culture medium from cells and replace with BrdU labeling solution.Incubate cells at 37°C for 2 hours.Remove labeling solution and wash two times with PBS.Wash with PBS (3 times, 2 minutes each)
Is EdU better than BrdU?
EdU staining with Click-iT technology, EdU vs BrdU assay. The simplicity of the click detection method makes the EdU assay a faster, friendlier alternative to the BrdU assay.
What is the difference between Ki67 and BrdU?
Ki67 and BrdU are two types of proliferation markers that are useful in the detection of cell proliferation. Ki67 is a specific protein, while BrdU is a synthetic nucleoside. Thus, this is the key difference between Ki67 and BrdU. Ki67 is able to label the cells in the G1, G2, S and M phases of the cell cycle.Sep 6, 2021
How do you do a BrdU assay?
Detect incorporated BrdURemove this solution and add 1 mL of antibody staining buffer.Add anti-BrdU primary antibody.Incubate overnight at room temperature.Wash with Triton X-100 permeabilization buffer (3 times, 2 minutes each)Add fluorescently labeled secondary antibody.Incubate one hour at room temperature.
What does a BrdU assay show?
Bromodeoxyuridine (BrdU) incorporation assays have long been used to detect DNA synthesis in vivo and in vitro. The key principle of this method is that BrdU incorporated as a thymidine analog into nuclear DNA represents a label that can be tracked using antibody probes.
What causes proliferation?
Cell proliferation leads to an exponential increase in cell number and is therefore a rapid mechanism of tissue growth. Cell proliferation requires both cell growth and cell division to occur at the same time, such that the average size of cells remains constant in the population.
Is BrdU an antibody?
BrdU is a thymidine analogue and when offered to proliferating cells it is incroporated into reduplicating cells. The antibody is specific for DNA in which BrdU has been incorporated. In immunoassays this antibody reacts strongly with free or carrier-protein coupled BrdU but not with other nucleosides.
What does EdU stain for?
5-Ethynyl-2′-deoxyuridine (EdU) is a thymidine analogue which is incorporated into the DNA of dividing cells. EdU is used to assay DNA synthesis in cell culture and detect cells in embryonic, neonatal and adult animals which have undergone DNA synthesis.
What is EdU staining used for?
Haematoxylin and eosin staining is frequently used in histology to examine thin tissue sections. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and other extracellular substances pink or red. Eosin is strongly absorbed by red blood cells, colouring them bright red.
How does EdU staining work?
In EdU staining, EdU is incorporated into newly synthesized DNA by cells within a sample. A fluorescent azide, such as iFluor-488, is then added. The fluorescent azide is small enough to diffuse freely through native tissues and DNA, and it covalently cross-links to the EdU in a 'click' chemistry reaction.
What is a BrdU?
BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells. BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. During the BrdU assay, BrdU is incorporated into replicating DNA ...
How long does it take for a cell to incubate in BrdU?
Note: BrdU incubation time depends on how rapidly the cells divide. Primary cells may need up to 24 hours, while rapidly proliferating cell lines may only need one hour. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.
How long to incubate cells in HCL?
Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour at room temperature. The exact HCl concentration and incubation time should be optimized for your experiment. If using a shorter incubation time, incubating at 37 o C may be more effective than room temperature.
What is the Ki67 protein?
See below for our suggestions. A cellular marker for proliferation, the Ki67 protein is present in cells at cycle phases G1, S, G2 and M, but absent in resting (G0) cells. Ki67 antibodies can be used instead of, or in conjunction with, BrdU to label proliferating neurons.