Bromodeoxyuridine (BrdU) is a thymidine analog and is specifically incorporated into DNA during DNA synthesis. Anti-bromodeoxyuridine antibody is used to identify cells that have incorporated BrdU. Anti-bromodeoxyuridine staining with flow cytometric analysis allows multiple parameters to be evaluated simultaneously. Anti-bromodeoxyuridine antibody has been used for identifying proliferating cells in blood, tissues , tumors, as well as for determining plasma cell labeling indices.
BrdU Labeling & ICC Protocol
Adherent or non-adherent cells can be labeled. Usually the labeling is done for a short period, such as two hours as shown in this example. However one can label overnight or even days depending upon the experiment.
1. Grow HUVEC cells in T flasks in traditional DMEM with 10% FBS.
2. Dissociate cells using trypsin/EDTA solution, and transfer to shell vials with 12 mm coverslips in 1 mL of appropriate media. (Alternatively polylysine-coated coverslips [0.05–0.1% in PBS,overnight using 150–300K mol. wt.] in 24 well plates or chamber-slides.)
3. Incubate cells at 37°C for 2 hours to allow for recovery.
4. Add BrdU* to each shell vial to a final concentration of 10 µM and incubate at 37°C for 2 hours.
5. Aspirate medium from the shell vials and wash the coverslips 3X in PBS.
6. Add 1 mL of Carnoy’s fixative (3 parts methanol :1 part glacial acetic acid) to each shell vial, aspirate out and immediately add an additional 1 mL of Carnoy’s fixative. (The first prewash with Carnoy’s ensures good fixation as it removes any traces of water from the previous washing step.) Fix shell vials at –20°C for 20 minutes.
7. Aspirate fixative from the shell vials and wash coverslips 3X in PBS.
8. Cells are denatured by adding 0.2 mL of 2M HCL in water to each shell vial and incubating at 37°C for 1 hour.
9. Aspirate acid from the shell vials and neutralize the coverslips by washing 3X in borate buffer pH 8.5.
10. Wash shell vials 3X in PBS + 0.05% Tween 20 [PBS/T20], and stain or cap and store at 2–8°C.
11. Cells are blocked by adding 0.2 mL of PBS/T20/2% normal goat serum to each vial and incubating at 37°C for 10–30 minutes
12. Aspirate out shell vials. Add 0.2 mL of anti-BrdU monoclonal antibody [cat. No. MAB3424, MAB4072], diluted in PBS/T20/2% normal goat serum. Incubate vials at 37°C for 30 minutes
Note: BrdU stock solution: 10 mM dissolved in PBS, 0.2 µm filter sterilized and stored at –20°C. BrdU labeling solution: BrdU stock solution was diluted 1/100 in culture medium to yield a 100 µM solution (10X). The 10X solution was added to each shell vial to a final concentration of 10 µM.
13. Wash shell vials 3X in PBS/T20, and add 0.2 mL of goat anti mouse FITC [cat. No. AP124F] conjugate in PBS/T20 to each vial [1:500–1:1000]. Incubate vials at 37°C for 30 minutes, in the dark.
14. Wash shell vials 3X in PBS/T20. Remove coverslips from the vials, dip in DI water and mount on glass slides, using mounting media for fluorescence [cat. No. 5013]. Prepared slides can be stored in a humid chamber, refrigerated and in the dark at least 2 hours to overnight.
15. Evaluate slides using fluorescence microscopy.