The incorporation of 5-bromo-2-deoxyuridine (BrdU) in replicating DNA of proliferating cells can be used to measure their proliferation. The following protocol allows the identification of cells which have incorporated BrdU by flow cytometry.
Method Article
5-bromo-2-deoxyuridine (BrdU) and 7-amino-actinomycin (7-AAD) staining for cell proliferation assay
https://doi.org/10.1038/nprot.2007.39
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The incorporation of 5-bromo-2-deoxyuridine (BrdU) in replicating DNA of proliferating cells can be used to measure their proliferation. The following protocol allows the identification of cells which have incorporated BrdU by flow cytometry.
BrdU (Roche)
Frosted microscope slides (Fisher Scientific)
RPMI-1640 medium (Cellgro)
ACK Lysing Buffer (Cambrex)
Cell strainers, 70 µm nylon (Becton Dickinson)
CD43 (Ly-48) magnetic beads and separation columns (Miltenyi)
0.5% Paraformaldehyde (Sigma) in PBS
3 N HCl/0.5% Tween 20 (freshly made, remember that concentrated HCl is 12 N)
0.1 M Disodium tetraborate (Sigma)
FACS buffer: PBS + 0.5% fetal calf serum (Sigma) + 5mM EDTA (Sigma)
FITC-labeled anti-BrdU (Pharmingen)
7-AAD solution (Pharmingen)
Injection of BrdU into mice
1 Inject 1 mg of BrdU, dissolved in 300 µl PBS, into the peritoneal cavity 12 h before analysis. Eventually, it is useful to start with injections 24 h before analysis and to inject a second time 12 h later.
Preparation and analysis of cells
▲Perform the following steps at 4 ˚C unless indicated otherwise.
2 Harvest splenocytes by grinding of spleens in RPMI-1640 medium between two frosted microscope slides. Spin cells at 1000 RPM for 5 min. Remove erythrocytes by adding one equivalent volume of ACK Lysing Buffer to the remaining pellet. Gently resuspend, swirl tube for 1 min. Fill tube with 10 ml of RPMI-1640, centrifuge at 1000 RPM for 5 min. Resuspend in 10 ml of RPMI-1640. Filter through 70 µm nylon cell strainer. Centrifuge at 1000 RPM for 5 min. Resuspend 1 x 107 cells in 500 µl of FACS buffer. Obtain B cells by CD43 negative purification using anti-CD43 magnetic beads.
3 Wash and resuspend 1×106 splenic B cells in 500 µl of 0.5% paraformaldehyde in PBS. Incubate on ice for 20 min (fixation). Spin and wash once with PBS.
4 Cell permeabilization: Spin and resuspend in 1 ml of freshly made 3 N HCl/0.5% Tween 20. Incubate at 25 ˚C for 20 min.
5 Spin and resuspend in 1 ml of 0.1 M disodium tetraborate. Spin and wash with FACS buffer.
6 Stain with FITC-labeled anti-BrdU. Incubate on ice for 20 min.
7 Spin and resuspend cells in 500 µl FACS buffer containing 2.0 µl of 7-AAD solution.
8 Analyze 10 min later.
▲NOTE: Because there is no dehydration step in this protocol, you still need to gate on the proper populations on forward scatter versus side scatter.
For preparation and analysis of cells, 2 h
Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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