brdu time course how to

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.

Full Answer

How long does it take for BrdU to work?

Note: BrdU incorporation into rapidly dividing tissues, such as the small intestine, will be detectable as soon as 30 minutes post-injection. However, for most tissue, you may need to wait up to 24 hours. The exact treatment time and dosage will need to be optimized for your tissue of interest.

How do you prepare BrdU solution?

Drinking water solutions of BrdU must be prepared in an appropriate chemical fume hood. Waste drinking water should be diluted to a 10% final solution of fresh mercury-free bleach, and may then be disposed in the drain. Water bottles should be washed with a 10% solution of fresh, mercury-free bleach.

How do you detect BrdU (BrdU)?

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 recommended dose of BrdU for animal studies?

Note: for mice, 225 mg/kg per day of BrdU (calculated by measuring water consumption volumes per animal) should achieve sufficient BrdU labeling. However, the exact dose should be optimized for individual experimental conditions.

How do you do a BrdU assay?

Prepare a 10 mM stock solution of BrdU (ab142567) by dissolving 3 mg of BrdU in 1 mL water. Dilute the 10 mM BrdU stock solution in cell culture medium to make a 10 µM BrdU labeling solution. Filter the 10 µM BrdU labeling solution through a 0.2 µm filter under sterile conditions.

How do you use BrdU?

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.

How do you dissolve BrdU?

For a 100X stock solution, prepare BrdU at 40 mg/mL in 10 mM Tris-Cl (pH 7.6) in a 15-mL screw-cap conical tube. Place the solution in a waterbath at ~50ºC and occasionally vortex vigorously until the BrdU is completely dissolved.

How does BrdU Labelling work?

Bromodeoxyuridine (BrdU) is a thymidine analog that incorporates DNA of dividing cells during the S-phase of the cell cycle. Once incorporated, these nucleoside analogs serve as cell cycle and proliferation markers that can be detected using labeled probes to identify cells that are actively proliferating.

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.

What is the difference between BrdU and EdU?

EdU assay. Unlike assays using BrdU staining, Click-iT EdU assays are not antibody-based and therefore do not require DNA denaturation for detection of the incorporated nucleoside.

How do you dissolve BrdU on PBS?

Dissolve BrDU (Sigma) in 7 mM NaOH/PBS to a final concentration of 16 mM (5 mg BrDU into 1 mL NaOH/PBS). Add the BrDU and shake, then heat to 37'C until in solution; cool before injecting. Perform an i.p. injection on pregnant females (~ 300 ul).

What is BrdU staining?

BrdU Staining and Labeling Protocols BrdU (5′-bromo-2′-deoxyuridine) is a thymidine analog that has been established as a popular tool in cancer and neuroscience research for determining cell proliferation rates.

What is BrdU assay?

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 is BrdU a marker of?

BrdU is not a marker of the S-phase of the cell cycle. As a thymidine analog, it is a marker of DNA synthesis.

Is BrdU radioactive?

Because BrdU can replace thymidine during DNA replication, it can cause mutations, and its use is therefore potentially a health hazard. However, because it is neither radioactive nor myelotoxic at labeling concentrations, it is widely preferred for in vivo studies of cancer cell proliferation.

What color is BrdU?

BrdU Cell Proliferation Assay Kit 6813Product IncludesQuantity (with Count)Solution ColorBrdU1 x 150 µlFixing/denaturing Solution2 x 25 mlBrdU Mouse Detection mAb1 x 500 µlGreenAnti-mouse IgG, HRP-Linked Antibody1 x 500 µlRed5 more rows

What was the goal of BrdU treatment?

BrdU is commonly used in the detection of proliferating cells in living tissues or as a cell cycle marker. It is administered to animals either shortly before euthanasia or as a chronic treatment. A common reason for chronic administration is to monitor the proliferation of experimentally administered stem cells.

Is BrdU hazardous?

BrdU is considered hazardous as it has mutagenic and strong teratogenic properties. This agent disrupts the normal activity of the cell. It is a suspected carcinogen and therefore exposure should be kept as low as reasonably achievable.

What is BrdU proliferation assay?

General description. BrdU Cell Proliferation Assay is a non-isotopic immunoassay for quantification of BrdU incorporation into newly synthesized DNA of actively proliferating cells. It is sensitive, rapid, and easy to perform.

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.

Select the Right BrdU Antibody for Your Experiment

Fig. 1. Jurkat cells were treated with 100 μM BrdU and cells were stained with clone RF04-2 Rat Anti-BrdU Antibody (MCA6143). As a secondary antibody, Rabbit F (ab')2 Anti-Rat IgG: DyLight488 Conjugated Antibody ( STAR16D488GA) was used.

Know Your Anti-BrdU Antibody Cross-Reactivity

BrdU and thymidine are structurally similar. Their structural difference is illustrated in Figure 2; in the case of BrdU, the 5-methyl group (shown in blue) of thymidine has been substituted by bromine (highlighted in green) (Kolb et al. 1999, Sivakumar et al. 2004).

Keep Your BrdU Fresh

Add freshly prepared BrdU to appropriate growth media for your cells or tissue. Remember that the half-life of BrdU at 4°C is short, so make sure your BrdU stock solution is stored at -20°C.

Perform a BrdU Titration

The ideal concentration of BrdU and incubation time for optimal labeling depends on the rate of cell division, and needs to be optimized for your cells. Perform titration experiments to determine the concentration of BrdU that will give the best level of incorporation without inducing cytotoxicity.

Optimize Your DNA Denaturation Step

For successful staining, it is important to include a DNA denaturation step to allow the antibody access to the incorporated BrdU. One common approach uses depurination and cleavage of DNA by acids such as hydrochloric acid. Other treatments with copper ions, heat, nucleases, and sodium hydroxide have also been reported (Liboska et al.

Optimize Your Washing Steps

After the denaturation step, ensure you perform sufficient washing in an appropriate amount of wash buffer. Any remaining acid that is not washed away could denature your antibody and affect the ability to detect BrdU.

Carefully Select Your Secondary Antibody

If you are planning to detect your anti-BrdU antibody with a secondary antibody, rather than a directly conjugated anti-BrdU antibody, background staining may be reduced by using a secondary antibody raised in a different species to the target cells/tissues.

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.

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.

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

Thymidine and BrdU, Cell Proliferation Assay Summary

How do you know if your cultured cells are growing? Does your new cancer drug affect cell proliferation? What’s the effect of VEGF on endothelial cells? As you can tell, knowing how to perform cell proliferation assays is an absolutely essential skill for anyone in biology, biochemistry, or pharmaceutics.

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.

Using Thymidine vs. BrdU. Cell Proliferation Assay Tips and Tricks

Taking things with a grain of salt: Note that DNA replication can happen even when cells are not proliferating. 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.

Other Methods: EdU Cell Proliferation Assay

BrdU immunohistochemistry has the disadvantage that you need antibodies to detect it. Because of this, you need to disrupt the tissue you are staining. EdU is a new version of BrdU which has an azide functional group. This can then easily be detected with a “click” fluorophore.

Step by Step Guide to BrdU Cell Proliferation assay in vivo

Here is how you can label proliferating cells in a mouse Materials for BrdU Assay BrdU labeling reagent (Invitrogen, #000103) BrdU staining kit (Invitrogen, #933943) Opaque dark container for storing stained tissue PBS 16% Paraformaldehyde in Water (#47608, Sigma Aldrich) 4% Paraformaldehyde 1% Paraformaldehyde with 0.5 M EDTA, pH 8 [Demineralization solution] Histo-clear (#50-329-51 Fisher Scientific) Ethanol 100% Ethanol 95% Ethanol 70% Water Slides Coverslips 30% Hydrogen Peroxide (H 2 O 2) in Water Methanol Petri dish with wet paper towels and paperclips for humidifying tissue (see image).

Notes on this BrdU Cell Proliferation Assay Methodology

Demineralization finishes when the tissue is pliable and can be bent without breaking.