How can you measure cell proliferation with flow cytometry?

How can you measure cell proliferation with flow cytometry?

Approaches to assess cell proliferation by flow cytometry include: measuring DNA synthesis with Invitrogen Click-iT EdU Flow Cytometry Assay Kits or traditional bromodeoxyuridine (BrdU) staining, tracking generations of cell division with Invitrogen CellTrace Cell Proliferation Kits or carboxyfluorescein succinimidyl …

What cells does BrdU stain for?

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 is lymphocyte proliferation measured?

There are several methods available to measure cell proliferation rates. One method is to measure the overall metabolic activity inside a cell. Several dyes are available that can permeabilize a cell and react with certain enzymes and other factors and form a colored end-product which can be easily detected.

What are BrdU cells?

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

How does BrdU labeling 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.

How does BrdU cause mutation?

During S phase of the cell cycle (when DNA replication occurs), BrdU can be incorporated in place of thymidine in newly synthesized DNA molecules of dividing cells. Because BrdU can replace thymidine during DNA replication, it can cause mutations, and its use is therefore potentially a health hazard.

How do I check my proliferation?

Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.

What is BrdU and how does it detect mitosis?

Bromodeoxyuridine (BrdU) variously abbreviated as BrdU, BudR, and BrdUrd, is a synthetic thymidine analog that gets incorporated into the DNA of dividing cells during the S-phase of the cell cycle and has a long history of heavy use in molecular and cytokinetic studies (1).

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.

How are BrdU and Edu used for cell proliferation?

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 the same principle as radioactive thymidine.

How is BrdU staining and BrdU assay performed?

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 and can be detected using anti-BrdU antibodies.

How long is the incubation period for 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.

What should the concentration of BrdU be in PBS?

Dilute BrdU in PBS to make a sterile solution of 10 mg/mL. For mice, as a general rule, inject the BrdU solution to a concentration of 100 mg/kg. After treatment with BrdU, the animals can be sacrificed according to your lab’s approved procedures. Fix and process tissue according to standard immunohistochemistry (IHC) protocols.