how to do brdu assay time course 5 days

What is BrdU assay and how is it used?

One common assay set-up is to supplement the culture media of growing cells with BrdU. By doing so, replicating cells, during the S phase of the cell cycle, incorporate BrdU instead of thymidine into newly synthesized DNA.

How long do you incubate cells in BrdU?

Incubate the cells in the BrdU labeling solution for 1–24 hours at 37ºC in a CO 2 incubator. 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.

What is the best way to label A Brdu antibody?

BrdU antibodies can be used in conjunction with cell type markers such as Ki67, doublecortin, and NeuN to identify proliferating cells and newly differentiated neurons. BrdU labeling: in vitro labeling of cells. 1. Prepare a 10 mM stock solution of BrdU (ab142567) by dissolving 3 mg of BrdU in 1 mL water.

What are the advantages of BrdU for detecting cell proliferation?

The main advantage of using BrdU for detecting cell proliferation is that it can be used in conjunction with a wide range of other antibodies raised against neuronal and non-neuronal cell types in the brain and its sensitivity to detect proliferating cells compared to other immunohistological methods.

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What is the Ames test?

Ames test is a method for evaluating mutagenic effects of implant device, chemicals, and drug utilizing bacteria to detect carcinogens and mutagens. The advantage of Ames test is its simplicity, low cost and being a fast assay (Seifalian et al.).

What is cytotoxicity test?

Cytotoxicity assay is a test for analyzing the cytotoxic effects of the material and medical device on the living organism ( Rosengren et al., 2005 ). It was the earliest and simplest in vitro technique that was designed for biocompatibility evaluation of materials. Examples of biological endpoints used in cytotoxicity testing include:

How long to incubate Triton X-100?

Wash with PBS (3 times, 2 minutes each) Remove PBS and add 1 mL of Triton X-100 permeabilization buffer to each well. Incubate for 20 minutes at room temperature.

What antibody is used to measure cell proliferation?

Cell proliferation can be measured with the thymidine analog BrdU (5-bromo-2’-deoxyuridine) following its incorporation into newly synthesized DNA and its subsequent detection with an anti-BrdU antibody.

What is the principle of cell proliferation assays?

Principle of Cell proliferation assays with nucleotide analogs. 3 H-Thymidine is a radioactive version of the Thymine DNA base (thymine + the sugar backbone = thymidine). When cells are incubated with thymidine, they use the radiolabeled thymidine to synthesize DNA and incorporate it into their DNA backbone.

How long has radioactive thymidine been used?

Radioactive Thymidine cell proliferation assays have been used since for over 40 years to detect whether cells are growing.

Does thymidine proliferate DNA?

The principle is simple: cells will incorporate Thymidine into their DNA as they proliferate. However, dealing with radioactivity is painful and annoying, so new fluorescence-based, non-radioactive, BrdU and EdU cell proliferation assays have become the new mainstay technique. These molecules are both thymidine analogs and hence work using ...

Is thymidine a perfect cell proliferation assay?

For example, if you have damaged DNA (ie. DNA repair is taking place). So, Thymidine and BrdU assays are really DNA replication assays and not perfect cell proliferation assays. But, for the most part, they are the gold-standard when looking for cell proliferation.

Can beta particles penetrate tissue?

And the beta particles that are generated by this method cannot penetrate very deep into tissue. So, if you’re labeling tissue sections, make sure they are extremely thin! In these cases, BrdU is a great option because it penetrates deep into tissue and can be detected even from 50 um thick slices.

Is BrdU a thymidine analog?

Similarly, BrdU is a Thymidine analog that la cks the radioactivity from tritium and it is used identically to Thymidine. Just incubate cells in the presence of BrdU. However, unlike radioactive thymidine, BrdU is detected with Anti-BrdU antibodies. A quick summary picture is shown below.

What phase of the cell cycle does BrdU occur?

By doing so, replicating cells, during the S phase of the cell cycle, incorporate BrdU instead of thymidine into newly synthesized DNA. BrdU is characterized by a very similar chemical structure to thymidine.

Can a mouse clone be used to detect incorporated BrdU?

Incorporated BrdU can be readily detected with anti-BrdU antibodies, such as the Mouse Anti-BrdU Antibody, clone Bu20a ( MCA2483) and Rabbit Anti-BrdU Antibody ( AHP2405 ). However, for successful staining it is important to include a DNA denaturing step to allow the antibody access to the incorporated BrdU. Acids, such as hydrochloric acid, are frequently used for this purpose; although treatments with copper ions, heat, nucleases, and sodium hydroxide have also been reported (Liboska et al. 2012, Kennedy et al. 2000).

What is a BrdU?

Bromo-deoxyuridine (BrdU) is a thymidine analog that is used in cell proliferation studies. BrdU in culture is incorporated into the DNA during DNA synthesis. Cellular incorporation of BrdU can be detected by anti-BrdU specific antibodies following membrane permeabilization by flow cytometry or immunohistochemistry. The molecular weight of BrdU is 307.1.

Can you label cells with BrdU?

Prior to BrdU immunostaining to detect proliferating cells it is necessary to label the cells or tissue with BrdU. Labeling can either be done in vitro for cell cultures or performed in vivo for experimental animals. Both protocols are provided.

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 37oC may be more effective than room temperature.

What is the cellular marker for proliferation?

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.

Can BrdU be detected in vivo?

BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. BrdU is incorporated into replicating DNA and can be detected using anti-BrdU antibodies.

How does BrdU label work?

The BrdU label within the DNA gets diluted with every round of DNA duplication in the cell cycle where unlabeled nucleotides are added to the cDNA and the labeled chromosomes are randomly distributed to the daughter cells. However, it takes several divisions to erase the label below detection limit. That means the LRCs that you see can have divided ones, twice or even more within your 3 weeks chase. If you consider that proliferative activity decreases with age ( that is pretty solidly known), it follows that cells in younger animals on aberage proliferate more and therefore dilute their initial BrdU-label more that those in older animals.

Why are FCM and BrdU used more often?

The reason that BrdU and FCM (PI or CFSE for exemple) are used more often is because they have been around for a long time. Also, they allow you to get informations that your DNA quantification assay does not. For example:

Can a fluorescent dye be used to track cells?

Yes. Because BrdU intercalates into DNA it can lead to DNA damage and consequently to changes in cellular behaviour. For tracking cells long-term, your best bet is to use a fluorescent dye such as CFSE or SNARF that bind protein. With each cell division after labelling, the amount of stain remaining is halved allowing you to estimate the rate of proliferation over time. Labelling with either of the above reagents is totally straightforward, just be sure to wash away any unincorporated dye before proceeding with your experiments. Good luck!

Introduction

The measurement of cell proliferation is fundamental to the assessment of cell health, genotoxicity, and drug efficacy. Proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog that is incorporated into DNA and detected using radioactivity, antibodies, or click chemistry.

Protocol

Use Jurkat cells in complete RPMI/10%FBS media, separate into 5 flasks

What Is Brdu? - Chemical Structure and Use in Cell Proliferation Assays

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Detection of Incorporated BrdU with Anti-Brdu Antibodies

BrdU Synonyms

References