In Vitro Labelling with Bromodeoxyuridine
Bromodeoxyuridine (BrdU) is a thymidine analog that is used in cell proliferation studies. BrdU in culture is incorporated into 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.
Preparation and Storage
Preparations of BrdU for in vivo or in vitro use should contain NO PRESERVATIVES. As such they should be handled under aseptic conditions. As with any biologic agent, avoid multiple freeze-thaws.
Store product at -80°C prior to use or for long-term storage of stock solutions.
Useful concentrations of BrdU stock solution for injection or in vitro use are 0.5 ml of a 10 mg/ml BrdU solution diluted in 1X DPBS. The BrdU solution, once thawed, is stable at 4°C for 2 weeks.
Application Notes / Recommended Assay Procedure
In order to detect BrdU immunostaining in proliferating cells it is first necessary to label the cells or tissue with BrdU. Labeling is either done in vitro for cell cultures or performed in vivo.
In vitro labeling of primary cells in culture and/or cell lines with BrdU
There are many different protocols for in vitro BrdU labeling of cells – one method is summarized here. As with all protocols, the method used should be optimized for your specific assay system before proceeding with experimental conditions.
Incubation of both primary cell cultures (PBMC’s etc) or cell lines with BrdU at a final concentration of 10 μM (diluted in cell culture medium) is an effective method for labeling a wide variety of cell lines from many different origins (human, mouse, rat, etc).
To label cells in vitro, add 10 μl of a 1 mM BrdU working solution (dilute BrdU Stock Solution 1:30 in tissue culture media to yield 1 mM solution) directly to each ml of tissue culture media. For labeling cells in 96 well plates it is convenient to make a 1:300 dilution of stock and add 10 ul per well of a 96 well plate containing 100 ul media. The cell culture density, in either case, should not exceed 2 x 10e6 cells/ml. Cells are incubated for 1-24 hours. The incubation time with BrdU is dependent on the cell rate of cell cycle (exact time should be empirically determined to achieve the optimal signal-to-noise ratio for your system). For example, an effective length of time for pulsing a rapidly proliferating cell line (e.g., HL60 cells ) is 30-45 minutes, (i.e., when the cells are in the logarithmic phase of cell growth). For primary cultures, such as peripheral blood lymphocytes with no exogenous stimulation (mitogen, etc.), an incubation time of 24 hours may be required. Investigators should empirically determine the incubation time that is optimal for their particular experimental system. Cells from the same population / treatment that are not BrdU-labeled should always be used as the negative cell staining control. This needs to be done with each experiment performed.
In vivo labeling of cells with BrdU
Two commonly used methods for in vivo BrdU labeling of tissues and cells include the intraperitoneal injection (IP) of a BrdU-containing solution into mice and the feeding of mice with BrdU that is added to their drinking water.
a) IP Method
A sterile solution of 10 mg/ml BrdU Dulbecos PBS is ideal for in vivo use. Inject mice i.p. with 100-200 μl (1-2 mg) of BrdU solution. Incorporation of BrdU can be detected in small intestine, thymus and bone marrow in as little as 0.5 hr post-injection. BrdU can be detected in most of the tissues 24 hrs post-injection, but this may be too long for rapidly dividing tissue such as the small intestine. Optimize treatment times by performing a time course for the tissue of interest before performing experiments.
b) Drinking Water Method
Dilute BrdU to 0.8 mg/ml in the drinking water. The BrdU mixture should be prepared freshly and changed daily. Long-term ingestion of BrdU will have toxic and/or lethal effects. For longer term studies, feeding mice with BrdU for 9 consecutive days followed by a rest period and feeding with water may be effective. BrdU incorporation may be detected in these cells past 70 days.
NOTE: BrdU has an unpleasant taste – a convenient trick is to add a bit of sucrose to the drinking water if the animals are not drinking the water containing BrdU.